PCan065 Antibody Compositions and Methods of Use

ABSTRACT

The invention provides isolated anti-PCan065 antibodies that bind to PCan065. The invention also encompasses compositions comprising an anti-PCan065 antibody and a carrier. These compositions can be provided in an article of manufacture or a kit. Another aspect of the invention is an isolated nucleic acid encoding an anti-PCan065 antibody, as well as an expression vector comprising the isolated nucleic acid. Also provided are cells that produce the anti-PCan065 antibodies. The invention encompasses a method of producing the anti-PCan065 antibodies. Other aspects of the invention are a method of killing an PCan065-expressing cancer cell, comprising contacting the cancer cell with an anti-PCan065 antibody and a method of alleviating or treating an PCan065-expressing cancer in a mammal, comprising administering a therapeutically effective amount of the anti-PCan065 antibody to the mammal.

This patent application is a continuation of U.S. application Ser. No.12/904,709, filed Oct. 14, 2010, which is a continuation of U.S.application Ser. No. 11/696,442 filed Apr. 4, 2007, which claims thebenefit of priority from U.S. Provisional Application Ser. No.60/789,360, filed Apr. 4, 2006, teachings of each of which are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to anti-PCan065 antibody compositions andmethods of detecting ovarian, breast, colon, prostate, pancreatic orlung cancer and killing PCan065-expressing ovarian, breast, colon,prostate, pancreatic or lung cancer cells.

BACKGROUND OF THE INVENTION Ovarian Cancer

Cancer of the ovaries is the fourth-most common cause of cancer death inwomen in the United States, with more than 23,000 new cases and roughly14,000 deaths predicted for the year 2001. Shridhar, V. et al., CancerRes. 61(15): 5895-904 (2001); Memarzadeh, S. & Berek, J. S., J. Reprod.Med, 46(7): 621-29 (2001). The American Cancer Society (ACS) estimatesthat there will be about 25,580 new cases of ovarian cancer in 2004 andovarian cancer will cause about 16,090 deaths in the United States. ACSWebsite: cancer with the extension .org of the world wide web. Morewomen die annually from ovarian cancer than from all other gynecologicmalignancies combined. The incidence of ovarian cancer in the US isestimated to 14.2 per 100,000 women per year and 9 women per 100,000 dieevery year from ovarian cancer. In 2004, approximately 70-75% of newdiagnoses will be stage III and IV carcinoma with a predicted 5-yearsurvival of ˜15%. Jemal et al., Annual Report to the Nation on theStatus of Cancer, 1975-2001, with a Special Feature Regarding Survival.Cancer 2004; 101: 3-27. The incidence of ovarian cancer is of seriousconcern worldwide, with an estimated 191,000 new cases predictedannually. Runnebaum, I. B. & Stickeler, E., J. Cancer Res. Can. Oncol.127(2): 73-79 (2001). Unfortunately, women with ovarian cancer aretypically asymptomatic until the disease has metastasized. Becauseeffective screening for ovarian cancer is not available, roughly 70% ofwomen diagnosed have an advanced stage of the cancer with a five-yearsurvival rate of ˜25-30%. Memarzadeh, S. & Berek, J. S., supra; Nunns,D. et al., Obstet. Gynecol. Surv. 55(12): 746-51. Conversely, womendiagnosed with early stage ovarian cancer enjoy considerably highersurvival rates. Werness, B. A. & Eltabbakh, G. H., Int'l. J. Gynecol.Pathol. 20(1): 48-63 (2001). Although our understanding of the etiologyof ovarian cancer is incomplete, the results of extensive research inthis area point to a combination of age, genetics, reproductive, anddietary/environmental factors. Age is a key risk factor in thedevelopment of ovarian cancer: while the risk for developing ovariancancer before the age of 30 is slim, the incidence of ovarian cancerrises linearly between ages 30 to 50, increasing at a slower ratethereafter, with the highest incidence being among septagenarian women.Jeanne M. Schilder et al., Hereditary Ovarian Cancer: Clinical Syndromesand Management, in Ovarian Cancer 182 (Stephen C. Rubin & Gregory P.Sutton eds., 2d ed. 2001).

With respect to genetic factors, a family history of ovarian cancer isthe most significant risk factor in the development of the disease, withthat risk depending on the number of affected family members, the degreeof their relationship to the woman, and which particular first degreerelatives are affected by the disease. Id. Mutations in several geneshave been associated with ovarian cancer, including BRCA1 and BRCA2,both of which play a key role in the development of breast cancer, aswell as hMSH2 and hMLH1, both of which are associated with hereditarynon-polyposis colon cancer. Katherine Y. Look, Epidemiology, Etiology,and Screening of Ovarian Cancer, in Ovarian Cancer 169, 171-73 (StephenC. Rubin & Gregory P. Sutton eds., 2d ed. 2001). BRCA1, located onchromosome 17, and BRCA2, located on chromosome 13, are tumor suppressorgenes implicated in DNA repair; mutations in these genes are linked toroughly 10% of ovarian cancers. Id. at 171-72; Schilder et al., supra at185-86. hMSH2 and hMLH1 are associated with DNA mismatch repair, and arelocated on chromosomes 2 and 3, respectively; it has been reported thatroughly 3% of hereditary ovarian carcinomas are due to mutations inthese genes. Look, supra at 173; Schilder et al., supra at 184, 188-89.

Reproductive factors have also been associated with an increased orreduced risk of ovarian cancer. Late menopause, nulliparity, and earlyage at menarche have all been linked with an elevated risk of ovariancancer. Schilder et al., supra at 182. One theory hypothesizes thatthese factors increase the number of ovulatory cycles over the course ofa woman's life, leading to “incessant ovulation,” which is thought to bethe primary cause of mutations to the ovarian epithelium. Id.; Laura J.Havrilesky & Andrew Berchuck, Molecular Alterations in Sporadic OvarianCancer, in Ovarian Cancer 25 (Stephen C. Rubin & Gregory P. Sutton eds.,2d ed. 2001). The mutations may be explained by the fact that ovulationresults in the destruction and repair of that epithelium, necessitatingincreased cell division, thereby increasing the possibility that anundetected mutation will occur. Id. Support for this theory may be foundin the fact pregnancy, lactation, and the use of oral contraceptives,all of which suppress ovulation, confer a protective effect with respectto developing ovarian cancer. Id.

Among dietary/environmental factors, there would appear to be anassociation between high intake of animal fat or red meat and ovariancancer, while the antioxidant Vitamin A, which prevents free radicalformation and also assists in maintaining normal cellulardifferentiation, may offer a protective effect. Look, supra at 169.Reports have also associated asbestos and hydrous magnesium trisilicate(talc), the latter of which may be present in diaphragms and sanitarynapkins Id. at 169-70.

Current screening procedures for ovarian cancer, while of some utility,are quite limited in their diagnostic ability, a problem that isparticularly acute at early stages of cancer progression when thedisease is typically asymptomatic yet is most readily treated. Walter J.Burdette, Cancer: Etiology, Diagnosis, and Treatment 166 (1998);Memarzadeh & Berek, supra; Runnebaum & Stickeler, supra; Werness &Eltabbakh, supra. 0Commonly used screening tests include biannualrectovaginal pelvic examination, radioimmunoassay to detect the CA-125serum tumor marker, and transvaginal ultrasonography. Burdette, supra at166. Currently, CA-125 is the only clinically approved serum marker foruse in ovarian cancer. CA-125 is found elevated in the majority ofserous cancers, but is elevated in only half of those women with earlystage disease. The major clinical application of CA125 is in monitoringtreatment success or detection of recurrence in women undergoingtreatment for ovarian cancer. Markman M. The Oncologist; 2: 6-9 (1997).The use of CA125 as a screening marker is limited because it isfrequently elevated in women with benign diseases such as endometriosis.Hence, there is a critical need for novel serum markers that are moresensitive and specific for the detection of ovarian cancer when usedalone, or in combination with CA125. Bast R C. Et al., Early Detectionof Ovarian Cancer: Promise and Reality in Ovarian Cancer. CancerResearch and Treatment Vol 107 (Stack M S, Fishman, D A, eds., 2001).

Pelvic examination has failed to yield adequate numbers of earlydiagnoses, and the other methods are not sufficiently accurate. Id. Onestudy reported that only 15% of patients who suffered from ovariancancer were diagnosed with the disease at the time of their pelvicexamination. Look, supra at 174. Moreover, the CA-125 test is prone togiving false positives in pre-menopausal women and has been reported tobe of low predictive value in post-menopausal women. Id at 174-75.Although transvaginal ultrasonography is now the preferred procedure forscreening for ovarian cancer, it is unable to distinguish reliablybetween benign and malignant tumors, and also cannot locate primaryperitoneal malignancies or ovarian cancer if the ovary size is normal.Schilder et al., supra at 194-95. While genetic testing for mutations ofthe BRCA1, BRCA2, hMSH2, and hMLH1 genes is now available, these testsmay be too costly for some patients and may also yield false negative orindeterminate results. Schilder et al., supra at 191-94.

Additionally, current efforts focus on the identification of panels ofbiomarkers that can be used in combination. Bast R C Jr., J Clin Oncol2003; 21: 200-205. Currently, other markers being evaluated as potentialovarian serum markers which may serve as members of a multi-marker panelto improve detection of ovarian cancer are HE4; mesothelin; kallikrein5, 8, 10 and 11; and prostasin. Urban et al. Ovarian cancer screeningHematol Oncol Clin North Am. 2003 August; 17(4):989-1005; Hellstrom etal. The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma, CancerRes. 2003 Jul. 1; 63(13):3695-700; Ordonez, Application of mesothelinimmunostaining in tumor diagnosis, Am J Surg Pathol. 2003 November;27(11):1418-28; Diamandis E P et al., Cancer Research 2002; 62: 295-300;Yousef G M et al., Cancer Research 2003; 63: 3958-3965; Kishi T et al.,Cancer Research 2003; 63: 2771-2774; Luo L Y et al., Cancer Research2003; 63: 807-811; Mok S C et al., J Natl Cancer Inst 2001; 93 (19):1437-1439.

The staging of ovarian cancer, which is accomplished through surgicalexploration, is crucial in determining the course of treatment andmanagement of the disease. AJCC Cancer Staging Handbook 187 (Irvin D.Fleming et al. eds., 5th ed. 1998); Burdette, supra at 170; Memarzadeh &Berek, supra; Shridhar et al., supra. Staging is performed by referenceto the classification system developed by the International Federationof Gynecology and Obstetrics. David H. Moore, Primary SurgicalManagement of Early Epithelial Ovarian Carcinoma, in Ovarian Cancer 203(Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001); Fleming et al.eds., supra at 188. Stage I ovarian cancer is characterized by tumorgrowth that is limited to the ovaries and is comprised of threesubstages. Id. In substage IA, tumor growth is limited to one ovary,there is no tumor on the external surface of the ovary, the ovariancapsule is intact, and no malignant cells are present in ascites orperitoneal washings. Id. Substage IB is identical to A1, except thattumor growth is limited to both ovaries. Id. Substage IC refers to thepresence of tumor growth limited to one or both ovaries, and alsoincludes one or more of the following characteristics: capsule rupture,tumor growth on the surface of one or both ovaries, and malignant cellspresent in ascites or peritoneal washings. Id.

Stage II ovarian cancer refers to tumor growth involving one or bothovaries, along with pelvic extension. Id. Substage IIA involvesextension and/or implants on the uterus and/or fallopian tubes, with nomalignant cells in the ascites or peritoneal washings, while substageIIB involves extension into other pelvic organs and tissues, again withno malignant cells in the ascites or peritoneal washings. Id. SubstageIIC involves pelvic extension as in IIA or IIB, but with malignant cellsin the ascites or peritoneal washings. Id

Stage III ovarian cancer involves tumor growth in one or both ovaries,with peritoneal metastasis beyond the pelvis confirmed by microscopeand/or metastasis in the regional lymph nodes. Id. Substage IIIA ischaracterized by microscopic peritoneal metastasis outside the pelvis,with substage IIIB involving macroscopic peritoneal metastasis outsidethe pelvis 2 cm or less in greatest dimension. Id. Substage IIIC isidentical to IIIB, except that the metastasis is greater than 2 cm ingreatest dimension and may include regional lymph node metastasis. Id.Lastly, Stage IV refers to the presence distant metastasis, excludingperitoneal metastasis. Id.

While surgical staging is currently the benchmark for assessing themanagement and treatment of ovarian cancer, it suffers from considerabledrawbacks, including the invasiveness of the procedure, the potentialfor complications, as well as the potential for inaccuracy. Moore, supraat 206-208, 213. In view of these limitations, attention has turned todeveloping alternative staging methodologies through understandingdifferential gene expression in various stages of ovarian cancer and byobtaining various biomarkers to help better assess the progression ofthe disease. Vartiainen, J. et al., Int'l J. Cancer, 95(5): 313-16(2001); Shridhar et al. supra; Baekelandt, M. et al., J Clin. Oncol.18(22): 3775-81.

The treatment of ovarian cancer typically involves a multiprong attack,with surgical intervention serving as the foundation of treatment.Dennis S. Chi & William J Hoskins, Primary Surgical Management ofAdvanced Epithelial Ovarian Cancer, in Ovarian Cancer 241 (Stephen C.Rubin & Gregory P. Sutton eds., 2d ed. 2001). For example, in the caseof epithelial ovarian cancer, which accounts for ˜90% of cases ofovarian cancer, treatment typically consists of: (1) cytoreductivesurgery, including total abdominal hysterectomy, bilateralsalpingo-oophorectomy, omentectomy, and lymphadenectomy, followed by (2)adjuvant chemotherapy with paclitaxel and either cisplatin orcarboplatin. Eltabbakh, G. H. & Awtrey, C. S., Expert Op. Pharmacother.2(10): 109-24. Despite a clinical response rate of 80% to the adjuvanttherapy, most patients experience tumor recurrence within three years oftreatment. Id. Certain patients may undergo a second cytoreductivesurgery and/or second-line chemotherapy. Memarzadeh & Berek, supra.

From the foregoing, it is clear that procedures used for detecting,diagnosing, monitoring, staging, prognosticating, and preventing therecurrence of ovarian cancer are of critical importance to the outcomeof the patient. Moreover, current procedures, while helpful in each ofthese analyses, are limited by their specificity, sensitivity,invasiveness, and/or their cost. As such, highly specific and sensitiveprocedures that would operate by way of detecting novel markers incells, tissues, or bodily fluids, with minimal invasiveness and at areasonable cost, would be highly desirable.

Accordingly, there is a great need for more sensitive and accuratemethods for predicting whether a person is likely to develop ovariancancer, for diagnosing ovarian cancer, for monitoring the progression ofthe disease, for staging the ovarian cancer, for determining whether theovarian cancer has metastasized, and for imaging the ovarian cancer.There is also a need for better treatment of ovarian cancer.

Angiogenesis in Cancer

Growth and metastasis of solid tumors are also dependent onangiogenesis. Folkman, J., 1986, Cancer Research, 46, 467-473; Folkman,J., 1989, Journal of the National Cancer Institute, 82, 4-6. It has beenshown, for example, that tumors which enlarge to greater than 2 mm mustobtain their own blood supply and do so by inducing the growth of newcapillary blood vessels. Once these new blood vessels become embedded inthe tumor, they provide a means for tumor cells to enter the circulationand metastasize to distant sites such as liver, lung or bone. Weidner,N., et al., 1991, The New England Journal of Medicine, 324(1), 1-8.

Angiogenesis, defined as the growth or sprouting of new blood vesselsfrom existing vessels, is a complex process that primarily occurs duringembryonic development. The process is distinct from vasculogenesis, inthat the new endothelial cells lining the vessel arise fromproliferation of existing cells, rather than differentiating from stemcells. The process is invasive and dependent upon proteolysis of theextracellular matrix (ECM), migration of new endothelial cells, andsynthesis of new matrix components. Angiogenesis occurs duringembryogenic development of the circulatory system; however, in adulthumans, angiogenesis only occurs as a response to a pathologicalcondition (except during the reproductive cycle in women).

Under normal physiological conditions in adults, angiogenesis takesplace only in very restricted situations such as hair growth andwounding healing. Auerbach, W. and Auerbach, R., 1994, Pharmacol Ther.63(3):265-3 11; Ribatti et al., 1991, Haematologica 76(4):3 11-20;Risau, 1997, Nature 386(6626):67 1-4. Angiogenesis progresses by astimulus which results in the formation of a migrating column ofendothelial cells. Proteolytic activity is focused at the advancing tipof this “vascular sprout”, which breaks down the ECM sufficiently topermit the column of cells to infiltrate and migrate. Behind theadvancing front, the endothelial cells differentiate and begin to adhereto each other, thus forming a new basement membrane. The cells thencease proliferation and finally define a lumen for the new arteriole orcapillary.

Unregulated angiogenesis has gradually been recognized to be responsiblefor a wide range of disorders, including, but not limited to, cancer,cardiovascular disease, rheumatoid arthritis, psoriasis and diabeticretinopathy. Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999, Circulation99(13): 1653-5; Koch, 1998, Arthritis Rheum 41(6):951-62; Walsh, 1999,Rheumatology (Oxford) 38(2):103-12; Ware and Simons, 1997, Nat Med 3(2):158-64.

Of particular interest is the observation that angiogenesis is requiredby solid tumors for their growth and metastases. Folkman, 1986 supra;Folkman 1990, J Natl. Cancer Inst., 82(1) 4-6; Folkman, 1992, SeminCancer Biol 3(2):65-71; Zetter, 1998, Annu Rev Med 49:407-24. A tumorusually begins as a single aberrant cell which can proliferate only to asize of a few cubic millimeters due to the distance from availablecapillary beds, and it can stay ‘dormant’ without further growth anddissemination for a long period of time. Some tumor cells then switch tothe angiogenic phenotype to activate endothelial cells, whichproliferate and mature into new capillary blood vessels. These newlyformed blood vessels not only allow for continued growth of the primarytumor, but also for the dissemination and recolonization of metastatictumor cells. The precise mechanisms that control the angiogenic switchis not well understood, but it is believed that neovascularization oftumor mass results from the net balance of a multitude of angiogenesisstimulators and inhibitors Folkman, 1995, supra.

One of the most potent angiogenesis inhibitors is endostatin identifiedby O'Reilly and Folkman. O'Reilly et al., 1997, Cell 88(2):277-85;O'Reilly et al., 1994, Cell 79(2):3 15-28. Its discovery was based onthe phenomenon that certain primary tumors can inhibit the growth ofdistant metastases. O'Reilly and Folkman hypothesized that a primarytumor initiates angiogenesis by generating angiogenic stimulators inexcess of inhibitors. However, angiogenic inhibitors, by virtue of theirlonger half life in the circulation, reach the site of a secondary tumorin excess of the stimulators. The net result is the growth of primarytumor and inhibition of secondary tumor. Endostatin is one of a growinglist of such angiogenesis inhibitors produced by primary tumors. It is aproteolytic fragment of a larger protein: endostatin is a 20 kDafragment of collagen XVIII (amino acid H1132-K1315 in murine collagenXVIII). Endostatin has been shown to specifically inhibit endothelialcell proliferation in vitro and block angiogenesis in vivo. Moreimportantly, administration of endostatin to tumor-bearing mice leads tosignificant tumor regression, and no toxicity or drug resistance hasbeen observed even after multiple treatment cycles. Boehm et al., 1997,Nature 390(6658):404-407. The fact that endostatin targets geneticallystable endothelial cells and inhibits a variety of solid tumors makes ita very attractive candidate for anticancer therapy. Fidler and Ellis,1994, Cell 79(2):185-8; Gastl et al., 1997, Oncology 54(3):177-84;Hinsbergh et al., 1999, Ann Oncol 10 Suppl 4:60-3. In addition,angiogenesis inhibitors have been shown to be more effective whencombined with radiation and chemotherapeutic agents. Klement, 2000, J.Clin Invest, 105(8) R15-24. Browder, 2000, Cancer Res. 6-(7) 1878-86,Arap et al., 1998, Science 279(5349):377-80; Mauceri et al., 1998,Nature 394(6690):287-91.

As discussed above, each of the methods for diagnosing and stagingovarian, breast, colon, prostate, pancreatic or lung cancer is limitedby the technology employed. Accordingly, there is need for sensitivemolecular and cellular markers for the detection of ovarian, breast,colon, prostate, pancreatic or lung cancer. There is a need formolecular markers for the accurate staging, including clinical andpathological staging, of ovarian, breast, colon, prostate, pancreatic orlung cancers to optimize treatment methods. In addition, there is a needfor sensitive molecular and cellular markers to monitor the progress ofcancer treatments, including markers that can detect recurrence ofovarian, breast, colon, prostate, pancreatic or lung cancers followingremission.

The present invention provides alternative methods of treating ovarian,breast, colon, prostate, pancreatic or lung cancer that overcome thelimitations of conventional therapeutic methods as well as offeradditional advantages that will be apparent from the detaileddescription below.

SUMMARY OF THE INVENTION

This invention is directed to an isolated PCan065 antibody that binds toPCan065 on a mammalian cell. The invention is further directed to anisolated PCan065 antibody that internalizes upon binding to PCan065 on amammalian cell. The antibody may be a monoclonal antibody.Alternatively, the antibody is an antibody fragment or a chimeric or ahumanized antibody. The monoclonal antibody may be produced by ahybridoma selected from the group of hybridomas deposited under AmericanType Culture Collection comprising PCan065.A10.3.2 and PCan065.B2.2.1,respectively.

The antibody may compete for binding to the same epitope as the epitopebound by the monoclonal antibody produced by a hybridoma selected fromthe group of hybridomas deposited under the American Type CultureCollection comprising PCan065.A10.3.2 and PCan065.B2.2.1, respectively.

The invention is also directed to conjugated antibodies. They may beconjugated to a growth inhibitory agent or a cytotoxic agent. Thecytotoxic agent may be selected from the group consisting of toxins,antibiotics, radioactive isotopes and nucleolytic enzymes and toxins.Examples of toxins include, but are not limited to, maytansin,maytansinoids, saporin, gelonin, ricin or calicheamicin.

The mammalian cell may be a cancer cell. Preferably, the anti-PCan065monoclonal antibody that inhibits the growth of PCan065-expressingcancer cells.

The antibody may be produced in bacteria. Alternatively, the antibodymay be a humanized form of an anti-PCan065 antibody produced by ahybridoma selected from the group of hybridomas deposited with the ATCCcomprising PCan065.A10.3.2 and PCan065.B2.2.1.

Preferably, the cancer is selected from the group consisting of ovarian,colon, prostate, and lung cancer. The invention is also directed to amethod of producing the antibodies comprising culturing an appropriatecell and recovering the antibody from the cell culture.

The invention is also directed to compositions comprising the antibodiesand a carrier. The antibody may be conjugated to a cytotoxic agent. Thecytotoxic agent may be a radioactive isotope or other chemotherapeuticagent.

The invention is also directed to a method of killing anPCan065-expressing cancer cell, comprising contacting the cancer cellwith the antibodies of this invention, thereby killing the cancer cell.The cancer cell may be selected from the group consisting of ovarian,colon, prostate, and lung cancer cell.

The ovarian, colon, prostate or lung may be metastatic cancer. Thebreast cancer may be HER-2 negative breast cancer. The invention is alsodirected to a method of alleviating an PCan065-expressing cancer in amammal, comprising administering a therapeutically effective amount ofthe antibodies to the mammal.

In addition, the invention is directed to an article of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises an antibody as described herein. The article ofmanufacture may also comprise an additional component, e.g., a packageinsert indicating that the composition can be used to treat ovarian,breast, colon, prostate, pancreatic or lung cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the PCan065 epitope map for anti-PCan065 antibodies.

FIG. 2 shows the PCan065 A10.3/B2.2 ELISA Standard Curve.

DETAILED DESCRIPTION OF THE INVENTION Definitions and General Techniques

Human “PCan065” as used herein, refers to a protein of 308 amino acidsthat is secreted by cells, whose nucleotide and amino acid sequencesequences are disclosed wholly or in part in e.g., WO 96/18730-A1 asHuman prostatic growth factor; JP07250688-A as Human TGF-betasuperfamily protein; WO 2001/81928-A1 as Human macrophage inhibitorycytokine 1 (MIC-1) wild type and allelic variant protein; WO2003/058021-A2 as Human apoptosis-associated protein SEQ ID 514; and WO97/00958-A1 as Human TGF-beta-like cytokine pCL13 and variants dd2, d2,h1, b1, u2, b2, a1, f1; the disclosures of which are hereby expresslyincorporated by reference. The 308 amino acid sequence of PCan065 isdepicted in SEQ ID NO:5. Amino acids 1-308, 30-308, 35-308, 197-308,207-308, 208-308 or 211-308 (with or without the N-terminal secretorysignal peptide and pre-, pro- and mature forms) of PCan065 are secretedfrom cells as single molecules, dimers or multi-mers. PCan065 as usedherein includes allelic variants and conservative substitution mutantsof the protein which have PCan065 biological activity.

PCan065 is related the Homo sapiens growth differentiation factor (GDF)family and is identified in the RefSeq database as accessionsNM_(—)004864 and NP_(—)004855 (accessible at ncbi with the extension.nlm.nih.gov of the world wide web) and titled “Homo sapiens growthdifferentiation factor 15 (GDF15)”. Other synonyms for PCan065 include:prostate differentiation factor (PDF); MIC1; PLAB; macrophage inhibitorycytokine (MIC-1); NAG-1; PTGF-beta (PTGFB); GDF-15; and NSAID(nonsteroidal anti-inflammatory drug)-activated protein 1. The refseqdatabase includes the following summary of PCan065:

-   -   Bone morphogenetic proteins (e.g., BMP5; MIM 112265) are members        of the transforming growth factor-beta (see TGFB1; MIM 190180)        superfamily and regulate tissue differentiation and maintenance.        They are synthesized as precursor molecules that are processed        at a dibasic cleavage site to release C-terminal domains        containing a characteristic motif of 7 conserved cysteines in        the mature protein. [supplied by OMIM]        Many publications have described the identification,        characterization, association with disease, and clinical        development of PCan065 as a molecular target for disease        detection, therapy and vaccination including the following which        are hereby incorporated by reference in their entirety.

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Serum macrophage inhibitory cytokine 1 as a marker ofpancreatic and other periampullary cancers. Clin Cancer Res. 2004 Apr 1;10(7): 2386-92. Baek SJ, Kim JS, Nixon JB, DiAugustine RP, Eling TE.Expression of NAG-1, a transforming growth factor-beta superfamilymember, by troglitazone requires the early growth response gene EGR-1. JBiol Chem. 2004 Feb 20; 279(8): 6883-92. Epub 2003 Dec 8. Tong S,Marjono B, Brown DA, Mulvey S, Breit SN, Manuelpillai U, Wallace EM.Serum concentrations of macrophage inhibitory cytokine 1 (MIC 1) as apredictor of miscarriage. Lancet. 2004 Jan 10; 363(9403): 129-30. KimKS, Shin JH, Baek SJ, Yoon JH. Expression of non-steroidalanti-inflammatory drug-activated gene-1 in human nasal mucosa andcultured nasal epithelial cells: a preliminary investigation. ActaOtolaryngol. 2003 Sep; 123(7): 857-61. Keelan JA, Wang K, ChaiworapongsaT, Romero R, Mitchell MD, Sato TA, Brown DA, Fairlie WD, Breit SN.Macrophage inhibitory cytokine 1 in fetal membranes and amniotic fluidfrom pregnancies with and without preterm labour and premature ruptureof membranes. Mol Hum Reprod. 2003 Sep; 9(9): 535-40. Liu T, Bauskin AR,Zaunders J, Brown DA, Pankhurst S, Russell PJ, Breit SN. Macrophageinhibitory cytokine 1 reduces cell adhesion and induces apoptosis inprostate cancer cells. Cancer Res. 2003 Aug 15; 63(16): 5034-40. Erratumin: Cancer Res. 2004 Jan 15; 64(2): 220. Panhurst, S [corrected toPankhurst, S]. Brown DA, Ward RL, Buckhaults P, Liu T, Romans KE,Hawkins NJ, Bauskin AR, Kinzler KW, Vogelstein B, Breit SN. MIC-1 serumlevel and genotype: associations with progress and prognosis ofcolorectal carcinoma. Clin Cancer Res. 2003 Jul; 9(7): 2642-50.Iczkowski KA, Pantazis CG. Overexpression of NSAID-activated geneproduct in prostate cancer. Int J Surg Pathol. 2003 Jul; 11(3): 159-66.Welsh JB, Sapinoso LM, Kern SG, Brown DA, Liu T, Bauskin AR, Ward RL,Hawkins NJ, Quinn DI, Russell PJ, Sutherland RL, Breit SN, Moskaluk CA,Frierson HF Jr, Hampton GM. Large-scale delineation of secreted proteinbiomarkers overexpressed in cancer tissue and serum. Proc Natl Acad SciUSA. 2003 Mar 18; 100(6): 3410-5. Epub 2003 Mar 6. Subramaniam S,Strelau J, Unsicker K. Growth differentiation factor-15 prevents lowpotassium-induced cell death of cerebellar granule neurons bydifferential regulation of Akt and ERK pathways. J Biol Chem. 2003 Mar14; 278(11): 8904-12. Epub 2003 Jan 3. Strausberg RL, Feingold EA,Grouse LH, Derge JG, Klausner RD, Collins FS, Wagner L, Shenmen CM,Schuler GD, Altschul SF, Zeeberg B, Buetow KH, Schaefer CF, Bhat NK,Hopkins RF, Jordan H, Moore T, Max SI, Wang J, Hsieh F, Diatchenko L,Marusina K, Farmer AA, Rubin GM, Hong L, Stapleton M, Soares MB, BonaldoMF, Casavant TL, Scheetz TE, Brownstein MJ, Usdin TB, Toshiyuki S,Carninci P, Prange C, Raha SS, Loquellano NA, Peters GJ, Abramson RD,Mullahy SJ, Bosak SA, McEwan PJ, McKernan KJ, Malek JA, Gunaratne PH,Richards S, Worley KC, Hale S, Garcia AM, Gay LJ, Hulyk SW, Villalon DK,Muzny DM, Sodergren EJ, Lu X, Gibbs RA, Fahey J, Helton E, Ketteman M,Madan A, Rodrigues S, Sanchez A, Whiting M, Madan A, Young AC,Shevchenko Y, Bouffard GG, Blakesley RW, Touchman JW, Green ED, DicksonMC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS,Krzywinski MI, Skalska U, Smailus DE, Schnerch A, Schein JE, Jones SJ,Marra MA; Mammalian Gene Collection Program Team. Generation and initialanalysis of more than 15,000 full-length human and mouse cDNA sequences.Proc Natl Acad Sci USA. 2002 Dec 24; 99(26): 16899-903. Epub 2002 Dec11. Wong J, Li PX, Klamut HJ. A novel p53 transcriptional repressorelement (p53TRE) and the asymmetrical contribution of two p53 bindingsites modulate the response of the placental transforming growthfactor-beta promoter to p53. J Biol Chem. 2002 Jul 19; 277(29):26699-707. Epub 2002 May 14. Graichen R, Liu D, Sun Y, Lee KO, Lobie PE.Autocrine human growth hormone inhibits placental transforming growthfactor-beta gene transcription to prevent apoptosis and allow cell cycleprogression of human mammary carcinoma cells. J Biol Chem. 2002 Jul 19;277(29): 26662-72. Epub 2002 May 6. Brown DA, Bauskin AR, Fairlie WD,Smith MD, Liu T, Xu N, Breit SN. Antibody-based approach to high-volumegenotyping for MIC-1 polymorphism. Biotechniques. 2002 Jul; 33(1):118-20, 122, 124 passim. Brown DA, Breit SN, Buring J, Fairlie WD,Bauskin AR, Liu T, Ridker PM. Concentration in plasma of macrophageinhibitory cytokine-1 and risk of cardiovascular events in women: anested case-control study. Lancet. 2002 Jun 22; 359(9324): 2159-63.Albertoni M, Shaw PH, Nozaki M, Godard S, Tenan M, Hamou MF, Fairlie DW,Breit SN, Paralkar VM, de Tribolet N, Van Meir EG, Hegi ME. Anoxiainduces macrophage inhibitory cytokine-1 (MIC-1) in glioblastoma cellsindependently of p53 and HIF-1. Oncogene. 2002 Jun 20; 21(27): 4212-9.Baek SJ, Wilson LC, Eling TE. Resveratrol enhances the expression ofnon-steroidal anti-inflammatory drug-activated gene (NAG-1) byincreasing the expression of p53. Carcinogenesis. 2002 Mar; 23(3):425-34. Buckhaults P, Rago C, St Croix B, Romans KE, Saha S, Zhang L,Vogelstein B, Kinzler KW. Secreted and cell surface genes expressed inbenign and malignant colorectal tumors. Cancer Res. 2001 Oct 1; 61(19):6996-7001. Baek SJ, Horowitz JM, Eling TE. Molecular cloning andcharacterization of human nonsteroidal anti-inflammatory drug-activatedgene promoter. Basal transcription is mediated by Sp1 and Sp3. J BiolChem. 2001 Sep 7; 276(36): 33384-92. Epub 2001 Jul 9. Fairlie WD, ZhangHP, Wu WM, Pankhurst SL, Bauskin AR, Russell PK, Brown PK, Breit SN. Thepropeptide of the transforming growth factor-beta superfamily member,macrophage inhibitory cytokine-1 (MIC-1), is a multifunctional domainthat can facilitate protein folding and secretion. J Biol Chem. 2001 May18; 276(20): 16911-8. Epub 2001 Feb 26. Baek SJ, Kim KS, Nixon JB,Wilson LC, Eling TE. Cyclooxygenase inhibitors regulate the expressionof a TGF-beta superfamily member that has proapoptotic andantitumorigenic activities. Mol Pharmacol. 2001 Apr; 59(4): 901-8.Fairlie WD, Russell PK, Wu WM, Moore AG, Zhang HP, Brown PK, Bauskin AR,Breit SN. Epitope mapping of the transforming growth factor-betasuperfamily protein, macrophage inhibitory cytokine-1 (MIC-1):identification of at least five distinct epitope specificities.Biochemistry. 2001 Jan 9; 40(1): 65-73. Moore AG, Brown DA, Fairlie WD,Bauskin AR, Brown PK, Munier ML, Russell PK, Salamonsen LA, Wallace EM,Breit SN. The transforming growth factor-ss superfamily cytokinemacrophage inhibitory cytokine-1 is present in high concentrations inthe serum of pregnant women. J Clin Endocrinol Metab. 2000 Dec; 85(12):4781-8. Strelau J, Sullivan A, Bottner M, Lingor P, Falkenstein E,Suter-Crazzolara C, Galter D, Jaszai J, Krieglstein K, Unsicker K.Growth/differentiation factor-15/macrophage inhibitory cytokine-1 is anovel trophic factor for midbrain dopaminergic neurons in vivo. JNeurosci. 2000 Dec 1; 20(23): 8597-603. Fairlie WD, Zhang H, Brown PK,Russell PK, Bauskin AR, Breit SN. Expression of a TGF-beta superfamilyprotein, macrophage inhibitory cytokine-1, in the yeast Pichia pastoris.Gene. 2000 Aug 22; 254(1-2): 67-76. Li PX, Wong J, Ayed A, Ngo D, BradeAM, Arrowsmith C, Austin RC, Klamut HJ. Placental transforming growthfactor-beta is a downstream mediator of the growth arrest and apoptoticresponse of tumor cells to DNA damage and p53 overexpression. J BiolChem. 2000 Jun 30; 275(26): 20127-35. Bauskin AR, Zhang HP, Fairlie WD,He XY, Russell PK, Moore AG, Brown DA, Stanley KK, Breit SN. Thepropeptide of macrophage inhibitory cytokine (MIC-1), a TGF-betasuperfamily member, acts as a quality control determinant for correctlyfolded MIC-1. EMBO J. 2000 May 15; 19(10): 2212-20. Fairlie WD, MooreAG, Bauskin AR, Russell PK, Zhang HP, Breit SN. MIC-1 is a novelTGF-beta superfamily cytokine associated with macrophage activation. JLeukoc Biol. 1999 Jan; 65(1): 2-5. Review. Bottner M, Laaff M,Schechinger B, Rappold G, Unsicker K, Suter-Crazzolara C.Characterization of the rat, mouse, and human genes ofgrowth/differentiation factor- 15/macrophage inhibiting cytokine-1(GDF-15/MIC-1). Gene. 1999 Sep 3; 237(1): 105-11. Paralkar VM, Vail AL,Grasser WA, Brown TA, Xu H, Vukicevic S, Ke HZ, Qi H, Owen TA, ThompsonDD. Cloning and characterization of a novel member of the transforminggrowth factor-beta/bone morphogenetic protein family. J Biol Chem. 1998May 29; 273(22): 13760-7. Lawton LN, Bonaldo MF, Jelenc PC, Qiu L,Baumes SA, Marcelino RA, de Jesus GM, Wellington S, Knowles JA,Warburton D, Brown S, Soares MB. Identification of a novel member of theTGF-beta superfamily highly expressed in human placenta. Gene. 1997 Dec5; 203(1): 17-26. Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG,Bansal M, He XY, Zhang HP, Donnellan M, Mahler S, Pryor K, Walsh BJ,Nicholson RC, Fairlie WD, Por SB, Robbins JM, Breit SN. MIC-1, a novelmacrophage inhibitory cytokine, is a divergent member of the TGF-betasuperfamily. Proc Natl Acad Sci USA. 1997 Oct 14; 94(21): 11514-9.Hromas R, Hufford M, Sutton J, Xu D, Li Y, Lu L. PLAB, a novel placentalbone morphogenetic protein. Biochim Biophys Acta. 1997 Oct 9; 1354(1):40-4. Yokoyama-Kobayashi M, Saeki M, Sekine S, Kato S. Human cDNAencoding a novel TGF-beta superfamily protein highly expressed inplacenta. J Biochem (Tokyo). 1997 Sep; 122(3): 622-6.

As described in the publications above, MIC-1/GDF-15 (PCan065) issecreted as a dimer with an approximate relative molecular mass of 24-30kDa. The 308 amino acid protein is characterized by N-terminal signalpeptide sequence from amino acids 1-29; an N-glycosylation site at aminoacid 70; a proteolytic cleavage site at amino acid 196; a disulfidebridge bond at amino acid 203, 211 or 273; and a transforming growthfactor-beta (TGF-beta) domain from amino acids 207-308, 208-308 or211-308. MIC-1 is expressed by cells as disulfide linked dimmer (Bausinet al. 2000, supra). It has been shown that PCan065/MIC-1 isdifferentially expressed in prostate, pancreatic and colon cancer versusnormal tissues. MIC-1 has cytokine activity to control the survival,growth, differentiation and effector function of tissues and cells; andgrowth factor activity that stimulates a cell to grow or proliferate.Additionally, MIC-1 plays a role in the regulation of a number ofprocesses including apoptosis, cell and growth differentiation, MAPkinase signaling, signal transduction, cell adhesion, lowpotassium-induced cell death of cerebellar granule neurons, p53associated expression, tumorigenesis and events such as miscarriage,pregnancy, and cardiovascular events. Specifically, TGF-beta is amultifunctional peptide that controls proliferation, differentiation,and other functions in many cell types.

Also described and known in the literature are antibodies againstMIC-1/GDF-15 such as sheep anti-MIC-1 polyclonal antibody 233B3 andmouse anti-MIC-1 monoclonal antibody 13C4H3 (Moore et al. 2000, supra);mouse anti-MIC-1 monoclonal antibody producing hybridomas 13, 26, 10, 14(Fairlie W D et al. 2001, supra); mouse anti-MIC-1 monoclonal antibody26G6H6 (Brown at al., Biotechniques 2002, supra); goat anti-MIC-1/GDF-15polyclonal antibody, catalogue No. AF957, from R&D Systems (Minneapolis,Minn.); and rabbit anti-MIC-1/GDF-15 polyclonal antibody, catalogue No.PAB-10692, from Orbigen (San Diego, Calif.). The disclosure,characterization and use of these antibodies and antibody producinghybridomas in their respective publications are herein incorporated byreference.

The antibodies of the instant invention specifically bind PCan065 andhave demonstrated characteristics which make them ideal therapeuticcandiates for modulating PCan065 activity or protein functions includingTGF-beta domain activity, cytokine activity, and growth factor activity.Modulation of these functions is achieved by binding of an antibody tothe functional domain and antagonisticly preventing the activity of thefunctional domain. Inhibition of PCan065 protein function may be alsoachevied by prevening or inhibiting activation of the PCan065pro-protein into the functional mature protien. Since conversion of thepro-protein to the mature protein is dependant on cleave of theproteolytic cleavage site, an anti-PCan065 antibody which binds to thecleavage site, or creates a steric block of the site preventingcleavage, would inhibit PCan065 maturation and reduce PCan065 proteinfunction. Alternatively, inhibition of PCan065 protein function may beachevied by disrupting, dissolving or preventing dimerization of PCan065with an anti-PCan065 antibody.

Inhibition of PCan065 protein function results in inhibition orreduction of PCan065 biological functions. Anti-PCan065 antibodies whichbind PCan065 inhibit or reduce PCan065 biological functions such asapoptosis, cell and growth differentiation, MAP kinase signaling, signaltransduction, cell adhesion, low potassium-induced cell death ofcerebellar granule neurons, p53 associated expression, tumorgenesis.

Furthermore, the antibodies of the instant invention are useful astherapeutic agents for individuals suffering from ovarian, breast,colon, prostate, pancreatic or lung cacrinomas. The antibodies may havetherapeutic effect by killing PCan065 expressing cancer cells,inhibiting growth of PCan065 expressing tumors, shrinking PCan065expressing tumors, extending survival time of individuals with PCan065expressing tumors, reducing metastases of PCan065 expressing tumors,inducing immune response against PCan065 expressing tumors, reducinginhibition of immune response against PCan065 expressing tumors orreducing angiogenesis or vascularization of PCan065 expressing tumors.

Taken together, the differential expression in cancer and role inregulation of cellular processes, make PCan065 a promising target fordiagnosis and immunotherapy of various tumor types. Anti-PCan065antibodies are useful in diagnostic or therapeutic applications alone orin combination with antibodies against other GDF family members.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g. bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. Preferably, the antibody will be purified (1)to greater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(VH) followed by three constant domains (CH) for each of the α and γchains and four CH domains for [L and F isotypes. Each 6 L chain has atthe N-terminus, a variable domain (VL) followed by a constant domain(CL) at its other end.

The VL is aligned with the VH and the CL is aligned with the firstconstant domain of the heavy chain (CHI).

Particular amino acid residues are believed to form an interface betweenthe light chain and heavy chain variable domains. The pairing of a VHand VL together forms a single antigen-binding site. For the structureand properties of the different classes of antibodies, see, e.g., Basicand Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Teff andTristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 1-10-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aP-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the P-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 5056 (L2) and 89-97 (L3) in the VL, and aroundabout 1-35 (H1), 50-65 (H2) and 95-102 (113) in the VH; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. residues 26-32 (L1),50-52 (L2) and 91-96 (U) in the VL, and 26-32 (HI), 53-55 (1-12) and96-101 (H3) in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917(1987)).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences•derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

An “intact” antibody is one which comprises an antigen-binding site aswell as a CL and at least heavy chain constant domains, CHI, CH2 andCH3. The constant domains may be native sequence constant domains (e.g.human native sequence constant domains) or amino acid sequence variantthereof. Preferably, the intact antibody has one or more effectorfunctions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments. Papain digestion of antibodies produces twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (VH), and the firstconstant domain of one heavy chain (CHI). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)2 fragment which roughly corresponds to two disulfide linkedFab fragments having divalent antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having additional few residues at the carboxy terminus ofthe CHI domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)2antibody fragments originally were produced as pairs of 8 Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the VH and VL domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the VH and VL domains of the twoantibodies are present on different polypeptide chains. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).Furthermore, effects of linker sequence alterations in engineeringbispecific tandem diabodies are described in Le Gall et al., Protein EngDes Sel. 17(4):357-66 (2004).

A “native sequence” polypeptide is one which has the same amino acidsequence as a polypeptide (e.g., antibody) derived from nature. Suchnative sequence polypeptides can be isolated from nature or can beproduced by recombinant or synthetic means. Thus, a native sequencepolypeptide can have the amino acid sequence of a naturally occurringhuman polypeptide, murine polypeptide, or polypeptide from any othermammalian species.

The term “amino acid sequence variant” refers to a polypeptide that hasamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants of PCan065 willpossess at least about 70% homology with the native sequence PCan065,preferably, at least about 80%, more preferably at least about 85%, evenmore preferably at least about 90% homology, and most preferably atleast 95%. The amino acid sequence variants can possess substitutions,deletions, insertions and/or alterations due to allelic variation orSingle Nucleotide Polymorphisms (SNPs) within the native nucleic acidsequence encoding the amino acid sequence.

Several definitions of SNPs exist. See, e.g., Brooks, 235 Gene 177-86(1999). As used herein, the term “single nucleotide polymorphism” or“SNP” includes all single base variants, thus including nucleotideinsertions and deletions in addition to single nucleotide substitutionsand any resulting amino acid variants due to codon alteration. There aretwo types of nucleotide substitutions. A transition is the replacementof one purine by another purine or one pyrimidine by another pyrimidine.A transversion is the replacement of a purine for a pyrimidine, or viceversa.

Numerous methods exist for detecting SNPs within a nucleotide sequence.A review of many of these methods can be found in Landegren et al., 8Genome Res. 769-76 (1998). For example, a SNP in a genomic sample can bedetected by preparing a Reduced Complexity Genome (RCG) from the genomicsample, then analyzing the RCG for the presence or absence of a SNP.See, e.g., WO 00/18960. Multiple SNPs in a population of targetpolynucleotides in parallel can be detected using, for example, themethods of WO 00/50869. Other SNP detection methods include the methodsof U.S. Pat. Nos. 6,297,018 and 6,322,980. Furthermore, SNPs can bedetected by restriction fragment length polymorphism (RFLP) analysis.See, e.g., U.S. Pat. Nos. 5,324,631; 5,645,995. RFLP analysis of SNPs,however, is limited to cases where the SNP either creates or destroys arestriction enzyme cleavage site. SNPs can also be detected by directsequencing of the nucleotide sequence of interest. In addition, numerousassays based on hybridization have also been developed to detect SNPsand mismatch distinction by polymerases and ligases. Several web sitesprovide information about SNPs including Ensembl (ensembl with theextension .org of the world wide web), Sanger Institute (sanger with theextension .ac.uk/genetics/exon/ of the world wide web), National Centerfor Biotechnology Information (NCBI) (ncbi with the extension .nlm.nih.gov/SNP/ of the world wide web), The SNP Consortium Ltd. (snp with theextension .cshl.org of the world wide web). The chromosomal locationsfor the compositions disclosed herein are provided below. In addition,one of ordinary skill in the art could perform a search against thegenome or any of the databases cited above using BLAST to find thechromosomal location or locations of SNPs. Another a preferred method tofind the genomic coordinates and associated SNPs would be to use theBLAT tool (genome.ucsc.edu, Kent et al. 2001, The Human Genome Browserat UCSC, Genome Research 996-1006 or Kent 2002 BLAT, The BLAST-LikeAlignment Tool Genome Research, 1-9). All web sites above were accessedDec. 3, 2003.

Preferred amino acid sequence variants of PCan065 are described in thetable below. The nucleic acid and amino acid sequences of PCan065 aredisclosed in the references cited above, which are incorporated byreference in their entirety. The polynucleotides encoding the aminoacids of the present invention were analyzed and single nucleotidepolymorphism (SNP) attributes were identified. Specifically identifiedwere SNPs occurring the coding region of the nucleotide, the Alleles ofthe SNP, the nucleotide ambiguity code for the SNP, the position in thecodon of the SNP if within the Open Reading Frame (1, 2, 3 or UTR foruntranslated regions), and the SNP type (synonymous or non-synonymous tothe protein translation). In addition to the attributes above, the SNPrs# ID for the NCBI SNP database (dbSNP) which is accessible at ncbiwith the extension .nlm.nih gov/SNP/ of the world wide web is referencedfor each SNP. Additional single nucleotide polymorphism (SNP)information can be accessed at the databases listed above.

The table below includes the polynucleotide target, dbSNP rs# ID,Nucleic acid residue affected by the SNP (Polynucleotide) inNM_(—)004864, SNP alleles, Nucleotide ambiguity code, Condon Position ofthe SNP if within the ORF (1, 2, 3 or UTR if not within ORF), and theSNP type (synonymous “syn” or non-synonymous “non-syn”), Amino acidresidue affected by the SNP (AA Residue) in NP_(—)003814, and theAlternate amino acid residue.

Nucleic Amino dbSNP Acid Ambiguity Codon SNP Acid Alternate rs# IDResidue Alleles Code Pos type Residue Amino Acid 1059519 50 C/G S 1Non-syn 9 L/V 17526133 50 C/G S 1 Non-syn 9 L/V 6413435 163 A/G R 3 Syn46 L/L 1059369 167 A/T W 1 Non-syn 48 T/S 17526140 167 A/T W 1 Non-syn48 T/S 17655466 167 A/T W 1 Non-syn 48 T/S 16982331 188 G/T K 1 Non-syn55 E/[stop] 1059022 358 T/C R 3 Syn 111 P/P 1804826 455 T/G K 3 Syn 140P/P 3746195 497 A/C M 1 Syn 158 R/R 1058587 629 G/C S 1 Non-syn 202 D/H1064601 770 G/C S 1 Non-syn 249 G/R 11556750 840 A/C M 2 Non-syn 272 H/P

Variants of PCan065 as described above and antibodies which bind tothese variants individually or in combination are part of the inventiondescribed herein. Antibodies of instant invention may have diagnostic orthereapeutic utility for the variants of PCan065 outlined above.

The phrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-IgEantibody is one which can bind to an IgE immunoglobulin in such a mannerso as to prevent or substantially reduce the ability of such moleculefrom having the ability to bind to the high affinity receptor, FcεRI.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. Sequence similarity may be measured by any common sequence analysisalgorithm, such as GAP or BESTFIT or other variation Smith-Watermanalignment. See, T. F. Smith and M. S. Waterman, J. Mol. Biol.147:195-197 (1981) and W.R. Pearson, Genomics 11:635-650 (1991).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

As used herein, an anti-PCan065 antibody that “internalizes” is one thatis taken up by (i.e., enters) the cell upon binding to PCan065 on amammalian cell (i.e. cell surface PCan065). The internalizing antibodywill of course include antibody fragments, human or humanized antibodyand antibody conjugate. For therapeutic applications, internalization invivo is contemplated. The number of antibody molecules internalized willbe sufficient or adequate to kill an PCan065-expressing cell, especiallyan PCan065-expressing cancer cell. Depending on the potency of theantibody or antibody conjugate, in some instances, the uptake of asingle antibody molecule into the cell is sufficient to kill the targetcell to which the antibody binds. For example, certain toxins are highlypotent in killing such that internalization of one molecule of the toxinconjugated to the antibody is sufficient to kill the tumor cell.

Whether an anti-PCan065 antibody internalizes upon binding PCan065 on amammalian cell can be determined by various assays including thosedescribed in the experimental examples below. For example, to testinternalization in vivo, the test antibody is labeled and introducedinto an animal known to have PCan065 expressed on the surface of certaincells. The antibody can be radiolabeled or labeled with fluorescent orgold particles, for instance. Animals suitable for this assay include amammal such as a NCR nude mouse that contains a human PCan065-expressingtumor transplant or xenograft, or a mouse into which cells transfectedwith human PCan065 have been introduced, or a transgenic mouseexpressing the human PCan065 transgene. Appropriate controls includeanimals that did not receive the test antibody or that received anunrelated antibody, and animals that received an antibody to anotherantigen on the cells of interest, which antibody is known to beinternalized upon binding to the antigen. The antibody can beadministered to the animal, e.g., by intravenous injection. At suitabletime intervals, tissue sections of the animal can be prepared usingknown methods or as described in the experimental examples below, andanalyzed by light microscopy or electron microscopy, for internalizationas well as the location of the internalized antibody in the cell. Forinternalization in vitro, the cells can be incubated in tissue culturedishes in the presence or absence of the relevant antibodies added tothe culture media and processed for microscopic analysis at desired timepoints. The presence of an internalized, labeled antibody in the cellscan be directly visualized by microscopy or by autoradiography ifradiolabeled antibody is used. Alternatively, in a quantitativebiochemical assay, a population of cells comprising PCan065-expressingcells are contacted in vitro or in vivo with a radiolabeled testantibody and the cells (if contacted in vivo, cells are then isolatedafter a suitable amount of time) are treated with a protease orsubjected to an acid wash to remove uninternalized antibody on the cellsurface. The cells are ground up and the amount of protease resistant,radioactive counts per minute (cpm) associated with each batch of cellsis measured by passing the homogenate through a scintillation counter.Based on the known specific activity of the radiolabeled antibody, thenumber of antibody molecules internalized per cell can be deduced fromthe scintillation counts of the ground-up cells. Cells are “contacted”with antibody in vitro preferably in solution form such as by adding thecells to the cell culture media in the culture dish or flask and mixingthe antibody well with the media to ensure uniform exposure of the cellsto the antibody. Instead of adding to the culture media, the cells canbe contacted with the test antibody in an isotonic solution such as PBSin a test tube for the desired time period. In vivo, the cells arecontacted with antibody by any suitable method of administering the testantibody such as the methods of administration described below whenadministered to a patient.

The faster the rate of internalization of the antibody upon binding tothe PCan065-expressing cell in vivo, the faster the desired killing orgrowth inhibitory effect on the target PCan065-expressing cell can beachieved, e.g., by a cytotoxic immunoconjugate. Preferably, the kineticsof internalization of the anti-PCan065 antibodies are such that theyfavor rapid killing of the PCan065-expressing target cell. Therefore, itis desirable that the anti-PCan065 antibody exhibit a rapid rate ofinternalization preferably, within 24 hours from administration of theantibody in vivo, more preferably within about 12 hours, even morepreferably within about 30 minutes to 1 hour, and most preferably,within about 30 minutes. The present invention provides antibodies thatinternalize as fast as about 15 minutes from the time of introducing theanti-PCan065 antibody in vivo. The antibody will preferably beinternalized into the cell within a few hours upon binding to PCan065 onthe cell surface, preferably within 1 hour, even more preferably within15-30 minutes. To determine if a test antibody can compete for bindingto the same epitope as the epitope bound by the anti-PCan065 antibodiesof the present invention including the antibodies produced by thehybridomas deposited with the ATCC, a cross-blocking assay e.g., acompetitive ELISA assay can be performed. In an exemplary competitiveELISA assay, PCan065-coated wells of a microtiter plate, orPCan065-coated SEPHAROSE® (high molecular weight substance for theseparation by gel filtration of macromolecules (such as viruses,proteins and blood serums) that range in weight from about 100,000 toseveral million (GE Healthcare Bio-Sciences Ab Ltd Liab Co Uppsala,Sweden) beads, are pre-incubated with or without candidate competingantibody and then a biotin-labeled anti-PCan065 antibody of theinvention is added. The amount of labeled anti-PCan065 antibody bound tothe PCan065 antigen in the wells or on the beads is measured usingavidin-peroxidase conjugate and appropriate substrate.

Alternatively, the anti-PCan065 antibody can be labeled, e.g., with aradioactive or fluorescent label or some other detectable and measurablelabel. The amount of labeled anti-PCan065 antibody that binds to theantigen will have an inverse correlation to the ability of the candidatecompeting antibody (test antibody) to compete for binding to the sameepitope on the antigen, i.e., the greater the affinity of the testantibody for the same epitope, the less labeled anti-PCan065 antibodywill be bound to the antigen-coated wells. A candidate competingantibody is considered an antibody that binds substantially to the sameepitope or that competes for binding to the same epitope as ananti-PCan065 antibody of the invention if the candidate competingantibody can block binding of the anti-PCan065 antibody by at least 20%,preferably by at least 20-50%, even more preferably, by at least 50% ascompared to a control performed in parallel in the absence of thecandidate competing antibody (but may be in the presence of a knownnoncompeting antibody). It will be understood that variations of thisassay can be performed to arrive at the same quantitative value.

An antibody having a “biological characteristic” of a designatedantibody, such as any of the monoclonal antibodies PCan065.A4,PCan065.A10, PCan065.A13, PCan065.B1, PCan065.B2, PCan065.B3,PCan065.B4, PCan065.B5, PCan065.B6, PCan065.B7, PCan065.B8, PCan065.B9,PCan065.B10, PCan065.B11, PCan065.B12, PCan065.B13, PCan065.B14,PCan065.B15, PCan065.B16, PCan065.B17, PCan065.B18, PCan065.B19,PCan065.B20, PCan065.B21, PCan065.B22, PCan065.B23, PCan065.B24,PCan065.B25, PCan065.B26, PCan065.B27, PCan065.B28, PCan065.B29,PCan065.B30, PCan065.B31, PCan065.B32, PCan065.B33, PCan065.B34,PCan065.B35, PCan065.B36, PCan065.B37, PCan065.B38, PCan065.B39,PCan065.B40, PCan065.B41, PCan065.B42, PCan065.B43, PCan065.B44,PCan065.B45, PCan065.B46, PCan065.B47, PCan065.B48, PCan065.B101,PCan065.B102, PCan065.B103, PCan065.B104, PCan065.B105, PCan065.B106,PCan065.B107, PCan065.B108, PCan065.B109, PCan065.B110, PCan065.B111,PCan065.B112, PCan065.B113, PCan065.B114, PCan065.B115, PCan065.B116,PCan065.B117 and PCan065.B118, is one which possesses one or more of thebiological characteristics of that antibody which distinguish it fromother antibodies that bind to the same antigen, PCan065.A4, PCan065.A10,PCan065.A13, PCan065.B1, PCan065.B2, PCan065.B3, PCan065.B4, PCan065.B5,PCan065.B6, PCan065.B7, PCan065.B8, PCan065.B9, PCan065.B10,PCan065.B11, PCan065.B12, PCan065.B13, PCan065.B14, PCan065.B15,PCan065.B16, PCan065.B17, PCan065.B18, PCan065.B19, PCan065.B20,PCan065.B21, PCan065.B22, PCan065.B23, PCan065.B24, PCan065.B25,PCan065.B26, PCan065.B27, PCan065.B28, PCan065.B29, PCan065.B30,PCan065.B31, PCan065.B32, PCan065.B33, PCan065.B34, PCan065.B35,PCan065.B36, PCan065.B37, PCan065.B38, PCan065.B39, PCan065.B40,PCan065.B41, PCan065.B42, PCan065.B43, PCan065.B44, PCan065.B45,PCan065.B46, PCan065.B47, PCan065.B48, PCan065.B101, PCan065.B102,PCan065.B103, PCan065.B104, PCan065.B105, PCan065.B106, PCan065.B107,PCan065.B108, PCan065.B109, PCan065.B110, PCan065.B111, PCan065.B112,PCan065.B113, PCan065.B114, PCan065.B115, PCan065.B116, PCan065.B117 andPCan065.B118, will bind the same epitope as that bound by PCan065.A4,PCan065.A10, PCan065.A13, PCan065.B 1, PCan065.B2, PCan065.B3,PCan065.B4, PCan065.B5, PCan065.B6, PCan065.B7, PCan065.B8, PCan065.B9,PCan065.B10, PCan065.B11, PCan065.B12, PCan065.B13, PCan065.B14,PCan065.B15, PCan065.B16, PCan065.B17, PCan065.B18, PCan065.B19,PCan065.B20, PCan065.B21, PCan065.B22, PCan065.B23, PCan065.B24,PCan065.B25, PCan065.B26, PCan065.B27, PCan065.B28, PCan065.B29,PCan065.B30, PCan065.B31, PCan065.B32, PCan065.B33, PCan065.B34,PCan065.B35, PCan065.B36, PCan065.B37, PCan065.B38, PCan065.B39,PCan065.B40, PCan065.B41, PCan065.B42, PCan065.B43, PCan065.B44,PCan065.B45, PCan065.B46, PCan065.B47, PCan065.B48, PCan065.B101,PCan065.B102, PCan065.B103, PCan065.B104, PCan065.B105, PCan065.B106,PCan065.B107, PCan065.B108, PCan065.B109, PCan065.B110, PCan065.B111,PCan065.B112, PCan065.B113, PCan065.B114, PCan065.B115, PCan065.B116,PCan065.B117 and PCan065.B118, (e.g. which competes for binding orblocks binding of monoclonal antibody PCan065.A4, PCan065.A10,PCan065.A13, PCan065.B1, PCan065.B2, PCan065.B3, PCan065.B4, PCan065.B5,PCan065.B6, PCan065.B7, PCan065.B8, PCan065.B9, PCan065.B10,PCan065.B11, PCan065.B12, PCan065.B13, PCan065.B14, PCan065.B15,PCan065.B16, PCan065.B17, PCan065.B18, PCan065.B19, PCan065.B20,PCan065.B21, PCan065.B22, PCan065.B23, PCan065.B24, PCan065.B25,PCan065.B26, PCan065.B27, PCan065.B28, PCan065.B29, PCan065.B30,PCan065.B31, PCan065.B32, PCan065.B33, PCan065.B34, PCan065.B35,PCan065.B36, PCan065.B37, PCan065.B38, PCan065.B39, PCan065.B40,PCan065.B41, PCan065.B42, PCan065.B43, PCan065.B44, PCan065.B45,PCan065.B46, PCan065.B47, PCan065.B48, PCan065.B101, PCan065.B102,PCan065.B103, PCan065.B104, PCan065.B105, PCan065.B106, PCan065.B107,PCan065.B108, PCan065.B109, PCan065.B110, PCan065.B111, PCan065.B112,PCan065.B113, PCan065.B114, PCan065.B115, PCan065.B116, PCan065.B117 andPCan065.B118,), be able to target an PCan065-expressing tumor in vivoand may internalize upon binding to PCan065 on a mammalian cell in vivo.Likewise, an antibody with the biological characteristic of thePCan065.A4, PCan065.A10, PCan065.A13, PCan065.B1, PCan065.B2,PCan065.B3, PCan065.B4, PCan065.B5, PCan065.B6, PCan065.B7, PCan065.B8,PCan065.B9, PCan065.B10, PCan065.B11, PCan065.B12, PCan065.B13,PCan065.B14, PCan065.B15, PCan065.B16, PCan065.B17, PCan065.B18,PCan065.B19, PCan065.B20, PCan065.B21, PCan065.B22, PCan065.B23,PCan065.B24, PCan065.B25, PCan065.B26, PCan065.B27, PCan065.B28,PCan065.B29, PCan065.B30, PCan065.B31, PCan065.B32, PCan065.B33,PCan065.B34, PCan065.B35, PCan065.B36, PCan065.B37, PCan065.B38,PCan065.B39, PCan065.B40, PCan065.B41, PCan065.B42, PCan065.B43,PCan065.B44, PCan065.B45, PCan065.B46, PCan065.B47, PCan065.B48,PCan065.B101, PCan065.B102, PCan065.B103, PCan065.B104, PCan065.B105,PCan065.B106, PCan065.B107, PCan065.B108, PCan065.B109, PCan065.B110,PCan065.B111, PCan065.B112, PCan065.B113, PCan065.B114, PCan065.B115,PCan065.B116, PCan065.B117 and PCan065.B118 antibody will have the sameepitope binding, targeting, internalizing, tumor growth inhibitory andcytotoxic properties of the antibody.

The term “antagonist” antibody is used in the broadest sense, andincludes an antibody that partially or fully blocks, inhibits, orneutralizes a biological activity of a native PCan065 protein disclosedherein. Methods for identifying antagonists of an PCan065 polypeptidemay comprise contacting an PCan065 polypeptide or a cell expressingPCan065 on the cell surface, with a candidate antagonist antibody andmeasuring a detectable change in one or more biological activitiesnormally associated with the PCan065 polypeptide.

The term ‘agonistic” antibody is used in the broadest sense, andincludes an antibody the partially or fully promotes, activates, orincreases biological activity of PCan065. Additionally, an agonisticantibody may mimic an PCan065 binding partner (e.g. receptor or ligand)wherein binding of the PCan065 antibody has substantially the sameeffect on biologic activity of PCan065 as binding of the bindingpartner. Methods for identifying agonists of an PCan065 polypeptide maycomprise contacting an PCan065 polypeptide or a cell expressing PCan065on the cell surface, with a candidate agonistic antibody and measuring adetectable change in one or more biological activities normallyassociated with the PCan065 polypeptide.

An “antibody that inhibits the growth of tumor cells expressing PCan065”or a “growth inhibitory” antibody is one which binds to and results inmeasurable growth inhibition of cancer cells expressing oroverexpressing PCan065. Preferred growth inhibitory anti-PCan065antibodies inhibit growth of PCan065-expressing tumor cells (e.g.,ovarian, colon, prostate or lung cancer cells) by greater than 20%,preferably from about 20% to about 50%, and even more preferably, bygreater than 50% (e.g. from about 50% to about 100%) as compared to theappropriate control, the control typically being tumor cells not treatedwith the antibody being tested. Growth inhibition can be measured at anantibody concentration of about 0.1 to 30 pg/ml or about 0.5 nM to 200nM in cell culture, where the growth inhibition is determined 1-10 daysafter exposure of the tumor cells to the antibody. Growth inhibition oftumor cells in vivo can be determined in various ways such as isdescribed in the Experimental Examples section below. The antibody isgrowth inhibitory in vivo if administration of the anti-PCan065 antibodyat about 1 pg/kg to about 100 mg/kg body weight results in reduction intumor size or tumor cell proliferation within about 5 days to 3 monthsfrom the first administration of the antibody, preferably within about 5to 30 days.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is usually one which overexpresses PCan065. Preferably the cell isa tumor cell, e.g. an ovarian, colon, prostate, or lung cell. Variousmethods are available for evaluating the cellular events associated withapoptosis. For example, phosphatidyl serine (PS) translocation can bemeasured by annexin binding; DNA fragmentation can be evaluated throughDNA laddering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody which induces apoptosis is one which results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcells in an annexin binding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC), complementdependent cytotoxicity (CDC); phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor); and B cell activation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or U.S. Pat. No. 5,821,337 may be performed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRI1B contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126.330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer, ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g. from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996) may be performed.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer ofthe urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,melanoma, multiple myeloma and B-cell lymphoma, brain, as well as headand neck cancer, and associated metastases.

A “PCan065-expressing cell” is a cell which expresses endogenous ortransfected PCan065 on the cell surface or secretes endogenous ortransfected PCan065. A “PCan065-expressing cancer” is a cancercomprising cells that have PCan065 protein present on the cell surfaceor secretes PCan065 from the cell. A “PCan065-expressing cancer”produces sufficient levels of PCan065 on the surface of cells thereof orsecretes PCan065 from the cells thereof, such that an anti-PCan065antibody can bind thereto and have a therapeutic effect with respect tothe cancer. A cancer which “overexpresses” PCan065 is one which hassignificantly higher levels of PCan065 at the cell surface thereof orsecretes PCan065 from the cells thereof, compared to a noncancerous cellof the same tissue type. Such overexpression may be caused by geneamplification or by increased transcription or translation. PCan065overexpression may be determined in a diagnostic or prognostic assay byevaluating increased levels of the PCan065 protein present on thesurface of a cell (e.g. via an immunohistochemistry assay; FACSanalysis). Alternatively, or additionally, one may measure levels ofPCan065-encoding nucleic acid or mRNA in the cell, e.g. via fluorescentin situ hybridization; (FISH; see W098/45479 published October, 1998),Southern blotting, Northern blotting, or polymerase chain reaction (PCR)techniques, such as real time quantitative PCR (RT-PCR). One may alsostudy PCan065 overexpression by measuring shed antigen in a biologicalfluid such as serum, e.g., using antibody-based assays (see also, e.g.,U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; W091/05264 published Apr.18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et al.J. Immunol. Methods 132: 73-80 (1990)). Aside from the above assays,various in vivo assays are available to the skilled practitioner. Forexample, one may expose cells within the body of the patient to anantibody which is optionally labeled with a detectable label, e.g. aradioactive isotope, and binding of the antibody to cells in the patientcan be evaluated, e.g. by external scanning for radioactivity or byanalyzing a biopsy taken from a patient previously exposed to theantibody. An PCan065-expressing cancer includes ovarian, breast, colon,prostate, pancreatic or lung cancer.

A “mammal” for purposes of treating a cancer or alleviating the symptomsof cancer, refers to any mammal, including-humans, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, cats, cattle,horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal ishuman.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for an PCan065-expressing cancer if, after receiving atherapeutic amount of an anti-PCan065 antibody according to the methodsof the present invention, the patient shows observable and/or measurablereduction in or absence of one or more of the following: reduction inthe number of cancer cells or absence of the cancer cells; reduction inthe tumor size; inhibition (i.e., slow to some extent and preferablystop) of cancer cell infiltration into peripheral organs including thespread of cancer into soft tissue and bone; inhibition (i.e., slow tosome extent and preferably stop) of tumor metastasis; inhibition, tosome extent, of tumor growth; and/or relief to some extent, one or moreof the symptoms associated with the specific cancer; reduced morbidityand mortality, and improvement in quality of life issues. To the extentthe anti-PCan065 antibody may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic. Reduction of these signsor symptoms may also be felt by the patient.

The above parameters for assessing successful treatment and improvementin the disease are readily measurable by routine procedures familiar toa physician. For cancer therapy, efficacy can be measured, for example,by assessing the time to disease progression (TTP) and/or determiningthe response rate (RR).

The term “therapeutically effective amount” refers to an amount of anantibody or a drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. See preceding definition of“treating”. To the extent the drug may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed.

Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, e.g., gelonin,ricin, saporin, and the various antitumor or anticancer agents disclosedbelow. Other cytotoxic agents are described below. A tumoricidal agentcauses destruction of tumor cells.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially anPCan065-expressing cancer cell, either in vitro or in vivo. Thus, thegrowth inhibitory agent may be one which significantly reduces thepercentage of PCan065-expressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce G1 arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), taxanes, and topoisomerase II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest GI also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WBSaunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxeland docetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Label” as used herein refers to a detectable compound or compositionwhich is conjugated directly or indirectly to the antibody so as togenerate a “labeled” antibody. The label may be detectable by itself(e.g. radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

The term “epitope tagged” used herein refers to a chimeric polypeptidecomprising an anti-PCan065 antibody polypeptide fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the Ig polypeptide to whichit is fused. The tag polypeptide is also preferably fairly unique sothat the antibody does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least six aminoacid residues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

An “isolated nucleic acid molecule” is a nucleic acid molecule, e.g., anRNA, DNA, or a mixed polymer, which is substantially separated fromother genome DNA sequences as well as proteins or complexes such asribosomes and polymerases, which naturally accompany a native sequence.The term embraces a nucleic acid molecule which has been removed fromits naturally occurring environment, and includes recombinant or clonedDNA isolates and chemically synthesized analogues or analoguesbiologically synthesized by heterologous systems. A substantially purenucleic acid molecule includes isolated forms of the nucleic acidmolecule.

“Vector” includes shuttle and expression vectors and includes, e.g., aplasmid, cosmid, or phagemid. Typically, a plasmid construct will alsoinclude an origin of replication (e.g., the ColEl origin of replication)and a selectable marker (e.g., ampicillin or tetracycline resistance),for replication and selection, respectively, of the plasmids inbacteria. An “expression vector” refers to a vector that contains thenecessary control sequences or regulatory elements for expression of theantibodies including antibody fragment of the invention, in prokaryotic,e.g., bacterial, or eukaryotic cells. Suitable vectors are disclosedbelow.

The cell that produces an anti-PCan065 antibody of the invention willinclude the parent hybridoma cell e.g., the hybridomas that aredeposited with the ATCC, as well as bacterial and eukaryotic host cellsinto which nucleic acid encoding the antibodies have been introduced.Suitable host cells are disclosed below.

RNA interference refers to the process of sequence-specific posttranscriptional gene silencing in animals mediated by short interferingRNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The correspondingprocess in plants is commonly referred to as post transcriptional genesilencing or RNA silencing and is also referred to as quelling in fungi.The process of post transcriptional gene silencing is thought to be anevolutionarily conserved cellular defense mechanism used to prevent theexpression of foreign genes which is commonly shared by diverse floraand phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protectionfrom foreign gene expression may have evolved in response to theproduction of double stranded RNAs (dsRNA) derived from viral infectionor the random integration of transposon elements into a host genome viaa cellular response that specifically destroys homologous singlestranded RNA or viral genomic RNA. The presence of dsRNA in cellstriggers the RNAi response though a mechanism that has yet to be fullycharacterized. This mechanism appears to be different from theinterferon response that results from dsRNA mediated activation ofprotein kinase PKR and 2′,5′-oligoadenylate synthetase resulting innon-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from dicer activity are typically about21-23 nucleotides in length and comprise about 19 base pair duplexes.Dicer has also been implicated in the excision of 21 and 22 nucleotidesmall temporal RNAs (stRNA) from precursor RNA of conserved structurethat are implicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex containing a siRNA, commonly referred to as an RNA-inducedsilencing complex (RISC), which mediates cleavage of single stranded RNAhaving sequence complementary to the antisense strand of the siRNAduplex. Cleavage of the target RNA takes place in the middle of theregion complementary to the antisense strand of the siRNA duplex(Elbashir et al., 2001, Genes Dev., 15, 188).

Short interfering RNA mediated RNAi has been studied in a variety ofsystems. Fire et al., 1998, Nature, 391, 806, were the first to observeRNAi in C. Elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70,describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,Nature, 404, 293, describe RNAi in Drosophila cells transfected withdsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced byintroduction of duplexes of synthetic 21-nucleotide RNAs in culturedmammalian cells including human embryonic kidney and HeLa cells. Recentwork in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J.,20, 6877) has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two nucleotide3′-overhangs. Furthermore, complete substitution of one or both siRNAstrands with 2′-deoxy(2′-H) or 2′-O-methyl nucleotides abolishes RNAiactivity, whereas substitution of the 3′-terminal siRNA overhangnucleotides with deoxynucleotides (2′-H) was shown to be tolerated.Single mismatch sequences in the center of the siRNA duplex were alsoshown to abolish RNAi activity. In addition, these studies also indicatethat the position of the cleavage site in the target RNA is defined bythe 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashiret al., 2001, EMBO J., 20, 6877). Other studies have indicated that a5′-phosphate on the target-complementary strand of a siRNA duplex isrequired for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-overhanging segments of a21-mer siRNA duplex having 2 nucleotide 3′ overhangs withdeoxyribonucleotides does not have an adverse effect on RNAi activity.Replacing up to 4 nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition,Elbashir et al., supra, also report that substitution of siRNA with2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al.,International PCT Publication No. WO 00/44914, and Beach et al.,International PCT Publication No. WO 01/68836 both suggest that siRNA“may include modifications to either the phosphate-sugar back bone orthe nucleoside to include at least one of a nitrogen or sulfurheteroatom”, however neither application teaches to what extent thesemodifications are tolerated in siRNA molecules nor provide any examplesof such modified siRNA. Kreutzer and Limmer, Canadian Patent ApplicationNo. 2,359,180, also describe certain chemical modifications for use indsRNA constructs in order to counteract activation of doublestranded-RNA-dependent protein kinase PKR, specifically 2′-amino or2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer and Limmer similarly fail to show towhat extent these modifications are tolerated in siRNA molecules nor dothey provide any examples of such modified siRNA.

Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that “RNAs with two (phosphorothioate) modified bases also hadsubstantial decreases in effectiveness as RNAi triggers (data notshown); (phosphorothioate) modification of more than two residuesgreatly destabilized the RNAs in vitro and we were not able to assayinterference activities.” Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and observed that substituting deoxynucleotides forribonucleotides “produced a substantial decrease in interferenceactivity”, especially in the case of Uridine to Thymidine and/orCytidine to deoxy-Cytidine substitutions. Id. In addition, the authorstested certain base modifications, including substituting 4-thiouracil,5-bromouracil, 5-iodouracil, 3-(aminoallyl)uracil for uracil, andinosine for guanosine in sense and antisense strands of the siRNA, andfound that whereas 4-thiouracil and 5-bromouracil were all welltolerated, inosine “produced a substantial decrease in interferenceactivity” when incorporated in either strand. Incorporation of5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resultedin substantial decrease in RNAi activity as well.

Beach et al., International PCT Publication No. WO 01/68836, describesspecific methods for attenuating gene expression using endogenouslyderived dsRNA. Tuschl et al., International PCT Publication No. WO01/75164, describes a Drosophila in vitro RNAi system and the use ofspecific siRNA molecules for certain functional genomic and certaintherapeutic applications; although Tuschl, 2001, Chem. Biochem., 2,239-245, doubts that RNAi can be used to cure genetic diseases or viralinfection due “to the danger of activating interferon response”. Li etal., International PCT Publication No. WO 00/44914, describes the use ofspecific dsRNAs for use in attenuating the expression of certain targetgenes. Zernicka-Goetz et al., International PCT Publication No. WO01/36646, describes certain methods for inhibiting the expression ofparticular genes in mammalian cells using certain dsRNA molecules. Fireet al., International PCT Publication No. WO 99/32619, describesparticular methods for introducing certain dsRNA molecules into cellsfor use in inhibiting gene expression. Plaetinck et al., InternationalPCT Publication No. WO 00/01846, describes certain methods foridentifying specific genes responsible for conferring a particularphenotype in a cell using specific dsRNA molecules. Mello et al.,International PCT Publication No. WO 01/29058, describes theidentification of specific genes involved in dsRNA mediated RNAi.Deschamps Depaillette et al., International PCT Publication No. WO99/07409, describes specific compositions consisting of particular dsRNAmolecules combined with certain anti-viral agents. Driscoll et al.,International PCT Publication No. WO 01/49844, describes specific DNAconstructs for use in facilitating gene silencing in targeted organisms.Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describes specificchemically modified siRNA constructs targeting the unc-22 gene of C.elegans. Tuschl et al., International PCT Publication No. WO 02/44321,describe certain synthetic siRNA constructs.

Compositions and Methods of the Invention

The invention provides anti-PCan065 antibodies. Preferably, theanti-PCan065 antibodies internalize upon binding to cell surface PCan065on a mammalian cell. The anti-PCan065 antibodies may also destroy orlead to the destruction of tumor cells expressing PCan065.

It was not apparent that PCan065 was internalization-competent. Inaddition the ability of an antibody to internalize depends on severalfactors including the affinity, avidity, and isotype of the antibody,and the epitope that it binds. We have demonstrated herein that the cellsurface PCan065 is internalization competent upon binding by theanti-PCan065 antibodies of the invention. Additionally, it wasdemonstrated that the anti-PCan065 antibodies of the present inventioncan specifically target PCan065-expressing tumor cells. These tumortargeting, internalization and growth inhibitory properties of theanti-PCan065 antibodies make these antibodies very suitable fortherapeutic uses, e.g., in the treatment of various cancers includingovarian, breast, colon, prostate, pancreatic or lung cancer.Internalization of the anti-PCan065 antibody is preferred, e.g., if theantibody or antibody conjugate has an intracellular site of action andif the cytotoxic agent conjugated to the antibody does not readily crossthe plasma membrane (e.g., the toxin calicheamicin). Internalization isnot necessary if the antibodies or the agent conjugated to theantibodies do not have intracellular sites of action, e.g., if theantibody can kill the tumor cell by ADCC or some other mechanism.

The anti-PCan065 antibodies of the invention also have variousnon-therapeutic applications. The anti-PCan065 antibodies of the presentinvention can be useful for diagnosis and staging of PCan065-expressingcancers (e.g., in radioimaging). They may be used alone or incombination with other ovarian cancer markers, including, but notlimited to, CA125, HE4 and mesothelin. The antibodies are also usefulfor purification or immunoprecipitation of PCan065 from cells, fordetection and quantitation of PCan065 in vitro, e.g. in an ELISA or aWestern blot, to kill and eliminate PCan065-expressing cells from apopulation of mixed cells as a step in the purification of other cells.The internalizing anti-PCan065 antibodies of the invention can be in thedifferent forms encompassed by the definition of “antibody” herein.Thus, the antibodies include full length or intact antibody, antibodyfragments, native sequence antibody or amino acid variants, humanized,chimeric or fusion antibodies, immunoconjugates, and functionalfragments thereof. In fusion antibodies, an antibody sequence is fusedto a heterologous polypeptide sequence. The antibodies can be modifiedin the Fc region to provide desired effector functions. As discussed inmore detail in the sections below, with the appropriate Fc regions, thenaked antibody bound on the cell surface can induce cytotoxicity, e.g.,via antibody-dependent cellular cytotoxicity (ADCC) or by recruitingcomplement in complement dependent cytotoxicity, or some othermechanism. Alternatively, where it is desirable to eliminate or reduceeffector function, so as to minimize side effects or therapeuticcomplications, certain other Fc regions may be used.

The antibody may compete for binding, or binds substantially to, thesame epitope bound by the antibodies of the invention. Antibodies havingthe biological characteristics of the present anti-PCan065 antibodies ofthe invention are also contemplated, e.g., an anti-PCan065 antibodywhich has the biological characteristics of a monoclonal antibodyproduced by the hybridomas deposited with the ATCC comprisingPCan065.A10.3.2 and PCan065.B2.2.1, specifically including the in vivotumor targeting, internalization and any cell proliferation inhibitionor cytotoxic characteristics. Specifically provided are anti-PCan065antibodies that bind to an epitope present in amino acids 1-10, 10-20,20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110,110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190,190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270,270-280, 280-290, 290-300, 301-308 or 1-15, 10-25, 15-25, 21-35, 31-45,41-55, 51-65, 61-75, 71-85, 81-95, 91-105, 101-115, 111-125, 121-135,131-145, 141-155, 151-165, 161-175, 171-185, 181-195, 191-205, 201-215,211-225, 221-235, 231-245, 241-255, 251-265, 261-275, 271-285, 281-295,291-300, 295-308 of human PCan065.

Methods of producing the above antibodies are described in detail below.

The present anti-PCan065 antibodies are useful for treating aPCan065-expressing cancer or alleviating one or more symptoms of thecancer in a mammal. Such a cancer includes ovarian, breast, colon,prostate, pancreatic or lung cancer, cancer of the urinary tract, lungcancer, breast cancer, colon cancer, pancreatic cancer, and ovariancancer, more specifically, prostate adenocarcinoma, renal cellcarcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lungsquamous cell carcinomas, and pleural mesothelioma. The cancersencompass metastatic cancers of any of the preceding, e.g., ovarian,breast, colon, prostate, pancreatic or lung cancer metastases. Theantibody is able to bind to at least a portion of the cancer cells thatexpress PCan065 in the mammal and preferably is one that does not induceor that minimizes HAMA response. Preferably, the antibody is effectiveto destroy or kill PCan065-expressing tumor cells or inhibit the growthof such tumor cells, in vitro or in vivo, upon binding to PCan065 on thecell. Such an antibody includes a naked anti-PCan065 antibody (notconjugated to any agent). Naked anti-PCan065 antibodies having tumorgrowth inhibition properties in vivo include the antibodies described inthe Experimental Examples below. Naked antibodies that have cytotoxic orcell growth inhibition properties can be further conjugated with acytotoxic agent to render them even more potent in tumor celldestruction. Cytotoxic properties can be conferred to an anti-PCan065antibody by, e.g., conjugating the antibody with a cytotoxic agent, toform an immunoconjugate as described below. The cytotoxic agent or agrowth inhibitory agent is preferably a small molecule. Toxins such asmaytansin, maytansinoids, saporin, gelonin, ricin or calicheamicin andanalogs or derivatives thereof, are preferable.

The invention provides a composition comprising an anti-PCan065 antibodyof the invention, and a carrier. For the purposes of treating cancer,compositions can be administered to the patient in need of suchtreatment, wherein the composition can comprise one or more anti-PCan065antibodies present as an immunoconjugate or as the naked antibody.Further, the compositions can comprise these antibodies in combinationwith other therapeutic agents such as cytotoxic or growth inhibitoryagents, including chemotherapeutic agents. The invention also providesformulations comprising an anti-PCan065 antibody of the invention, and acarrier. The formulation may be a therapeutic formulation comprising apharmaceutically acceptable carrier.

Another aspect of the invention is isolated nucleic acids encoding theinternalizing anti-PCan065 antibodies. Nucleic acids encoding both the Hand L chains and especially the hypervariable region residues, chainswhich encode the native sequence antibody as well as variants,modifications and humanized versions of the antibody, are encompassed.

The invention also provides methods useful for treating anPCan065-expressing cancer or alleviating one or more symptoms of thecancer in a mammal, comprising administering a therapeutically effectiveamount of an internalizing anti-PCan065 antibody to the mammal. Theantibody therapeutic compositions can be administered short term (acute)or chronic, or intermittent as directed by physician. Also provided aremethods of inhibiting the growth of, and killing an PCan065 expressingcell. Finally, the invention also provides kits and articles ofmanufacture comprising at least one antibody of this invention,preferably at least one internalizing anti-PCan065 antibody of thisinvention. Kits containing anti-PCan065 antibodies find use in detectingPCan065 expression, or in therapeutic or diagnostic assays, e.g., forPCan065 cell killing assays or for purification and/orimmunoprecipitation of PCan065 from cells, tissues or bodily fluids. Forexample, for isolation and purification of PCan065, the kit can containan anti-PCan065 antibody coupled to a solid support, e.g., a tissueculture plate or beads (e.g., SEPHAROSE® beads). Kits can be providedwhich contain antibodies for detection and quantitation of PCan065 invitro, e.g. in an ELISA or a Western blot. Such antibody useful fordetection may be provided with a label such as a fluorescent orradiolabel.

Production of Anti-PCan065 Antibodies

The following describes exemplary techniques for the production of theantibodies useful in the present invention. Some of these techniques aredescribed further in Example 1. The PCan065 antigen to be used forproduction of antibodies may be, e.g., the full length polypeptide or aportion thereof, including a soluble form of PCan065 lacking themembrane spanning sequence, or synthetic peptides to selected portionsof the protein.

Alternatively, cells expressing PCan065 at their cell surface (e.g. CHOor NIH-3T3 cells transformed to overexpress PCan065; ovarian,pancreatic, lung, breast or other PCan065-expressing tumor cell line),or membranes prepared from such cells can be used to generateantibodies. The nucleotide and amino acid sequences of human and murinePCan065 are available as provided above. PCan065 can be producedrecombinantly in and isolated from, prokaryotic cells, e.g., bacterialcells, or eukaryotic cells using standard recombinant DNA methodology.PCan065 can be expressed as a tagged (e.g., epitope tag) or other fusionprotein to facilitate its isolation as well as its identification invarious assays.

Antibodies or binding proteins that bind to various tags and fusionsequences are available as elaborated below. Other forms of PCan065useful for generating antibodies will be apparent to those skilled inthe art.

Tags

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990)). The FLAG-peptide (Hopp et al., BioTechnology, 6:1204-1210(1988)) is recognized by an anti-FLAG M2 monoclonal antibody (EastmanKodak Co., New Haven, Conn.). Purification of a protein containing theFLAG peptide can be performed by immunoaffinity chromatography using anaffinity matrix comprising the anti-FLAG M2 monoclonal antibodycovalently attached to agarose (Eastman Kodak Co., New Haven, Conn.).Other tag polypeptides include the KT3 epitope peptide [Martin et al.,Science, 255:192-194 (1992)]; an α-tubulin epitope peptide (Skinner etal., J. Biol. Chenz., 266:15163-15166 (1991)); and the T7 gene proteinpeptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)).

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals, preferablynon-human animals, by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the relevant antigen and an adjuvant. It may be useful toconjugate the relevant antigen (especially when synthetic peptides areused) to a protein that is immunogenic in the species to be immunized.For example, the antigen can be conjugated to keyhole limpet hemocyanin(KLH), serum, bovine thyroglobulin, or soybean trypsin inhibitor, usinga bifunctional or derivatizing agent, e.g., maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride, SOC1₂, or R¹ N═C═NR, where R and R¹ are different alkylgroups. Conjugates also can be made in recombinant cell culture asprotein fusions.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 5-100 pg of the protein or conjugate(for rabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later, the animals are boosted with ⅕ to 1/10 the original amountof peptide or conjugate in Freund's complete adjuvant by subcutaneousinjection at multiple sites. Seven to 14 days later, the animals arebled and the serum is assayed for antibody titer. Animals are boosteduntil the titer plateaus. Also, aggregating agents such as alum aresuitably used to enhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridomamethod, a mouse or other appropriate host animal, such as a hamster, isimmunized as described above to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. After immunization, lymphocytes are isolated andthen fused with a “fusion partner”, e.g., a myeloma cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, Monoclonal Antibodies. Principles and Practice, pp 103(Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, fusion partner,e.g., the parental myeloma cells. For example, if the parental myelomacells lack the enzyme hypoxanthine guanine phosphoribosyl transferase(HGPRT or HPRT), the selective culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (HATmedium), which substances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-IImouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Rockville, Md. USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980). Once hybridoma cells that produce antibodiesof the desired specificity, affinity, and/or activity are identified,the clones may be subcloned by limiting dilution procedures and grown bystandard methods (Goding, Monoclonal Antibodies: Principles andPractice, pp 103 (Academic Press, 1986)). Suitable culture media forthis purpose include, for example, D-MEM or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal e.g., by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-SEPHAROSE®)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transformed or transfected intoprokaryotic or eukaryotic host cells such as, e.g., E. coli cells,simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells,that do not otherwise produce antibody protein, to obtain the synthesisof monoclonal antibodies in the recombinant host cells. Review articleson recombinant expression in bacteria of DNA encoding the antibodyinclude Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) andPhickthun, Immunol. Revs., 130:151-188 (1992).

Further, the monoclonal antibodies or antibody fragments can be isolatedfrom antibody phage libraries generated using the techniques describedin McCafferty et al., Nature, 348:552-554 (1990). Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991) describe the isolation of murine and human antibodies,respectively, using phage libraries. Subsequent publications describethe production of high affinity (nM range) human antibodies by chainshuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (CH and CL) sequences for thehomologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenonimmunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is nonhuman. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.,151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art.

Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the hypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

Various forms of a humanized anti-PCan065 antibody are contemplated. Forexample, the humanized antibody may be an antibody fragment, such as aFab, which is optionally conjugated with one or more cytotoxic agent(s)in order to generate an immunoconjugate. Alternatively, the humanizedantibody may be an intact antibody, such as an intact IgG1 antibody.

Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); 5,545,807; and Alternatively, phage displaytechnology (McCafferty et al., Nature 348:552-553 (1990)) can be used toproduce human antibodies and antibody fragments in vitro, fromimmunoglobulin variable (V) domain gene repertoires from unimmunizeddonors. According to this technique, antibody V domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, such as Ml3 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B-cell.Phage display can be performed in a variety of formats, reviewed in,e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion inStructural Biology 3:564-571 (1993). Several sources of V-gene segmentscan be used for phage display. Clackson et al., Nature, 352:624-628(1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991),or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos.5,565,332 and 5,573,905. As discussed above, human antibodies may alsobe generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610and 5,229,275).

Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors. Various techniques have been developed for the productionof antibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992); andBrennan et al., Science, 229:81 (1985)). However, these fragments cannow be produced directly by recombinant host cells. Fab, Fv and ScFvantibody fragments can all be expressed in and secreted from E. coli,thus allowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab)₂ fragments(Carter et al., Bio/Technology 10: 163-167 (1992)). According to anotherapproach, F(ab)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. The antibody of choice may also be a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat.No. 5,587,458. Fv and sFv are the only species with intact combiningsites that are devoid of constant regions; thus, they are suitable forreduced nonspecific binding during in vivo use. sFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the PCan065 protein. Other suchantibodies may combine an PCan065 binding site with a binding site foranother protein. Alternatively, an anti-PCan065.Arm may be combined withan arm which binds to a triggering molecule on a leukocyte such as aTcell receptor molecule (e.g. C133), or Fc receptors for IgG (FcγR),such as FcγRI (CD64), FcγR1I (CD32) and FcγRIII (CD16), so as to focusand localize cellular defense mechanisms to the PCan065-expressing cell.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express PCan065. These antibodies possess an PCan065-bindingarm and an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab)₂ bispecificantibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIIIantibody and U.S. Pat. No. 5,837,234 discloses a bispecificanti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody isshown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecificanti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (CHI) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

Preferably, the bispecific antibodies in this approach are composed of ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. It was foundthat this asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e g tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agent,sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers.

The “diabody” technology described by Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternativemechanism for making bispecific antibody fragments. The fragmentscomprise a VH connected to a VL by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1(X1n-VD2-(X2)n-Fc,wherein VDI is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, XI and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CHI-flexible linker-VH-CHI-Fcregion chain; or VH-CHI-VH-CHI-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the anti-PCan065 antibodiesdescribed herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody Amino acid sequence variants of the anti-PCan065 antibody areprepared by introducing appropriate nucleotide changes into theanti-PCan065 antibody nucleic acid, or by peptide synthesis.

Such modifications include, for example, deletions from, and/orinsertions into, and/or substitutions of, residues within the amino acidsequences of the anti-PCan065 antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe anti-PCan065 antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theanti-PCan065 antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells in Science, 244:1081-1085 (1989). Here, a residue or group oftarget residues within the anti-PCan065 antibody are identified (e.g.,charged residues such as arg, asp, his, lys, and glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with PCan065antigen.

Those amino acid locations demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat, or for, the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, toanalyze the performance of a mutation at a given site, ala scanning orrandom mutagenesis is conducted at a target codon or region and theexpressed anti-PCan065 antibody variants are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-PCan065 antibody with an N-terminal methionyl residue or theantibody fused to a cytotoxic polypeptide. Other insertional variants ofthe anti-PCan065 antibody molecule include the fusion to the N- orC-terminus of the anti-PCan065 antibody to an enzyme (e.g. for ADEPT) ora fusion to a polypeptide which increases the serum half-life of theantibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-PCan065antibody molecule replaced by a different residue. The sites of greatestinterest for substitutional mutagenesis include the hypervariableregions, but FR alterations are also contemplated. Conservativesubstitutions are shown in Table I under the heading of “preferredsubstitutions”. If such substitutions result in a change in biologicalactivity, then more substantial changes, denominated “exemplarysubstitutions” in the table below, or as further described below inreference to amino acid classes, may be introduced and the productsscreened for a desired characteristic.

Amino Acid Substitutions

Original Preferred Residue Exemplary Substitutions Substitutions Ala (A)val; leu; ile Val Arg (R) lys; g1n; asn lys Asn (N) g1n; his; asp, lys;arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asnGlu (E) asp; gln asp Gly (G) ala ala His (H) asn; g1n; lys; arg arg Ile(I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile;val; met; ala; phe ile Lys (K) arg; gin; asn arg Met (M) leu; phe; ileleu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thrThr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser Phe Val(V) ile; leu; met; phe; ala; norleucine leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutralhydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin,his, lys, arg; (5) residues that influence chain orientation: gly, pro;and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of the anti-PCan065 antibody alsomay be substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond(s) may be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino acid substitutions at each site. Theantibody variants thus generated are displayed in a monovalent fashionfrom filamentous phage particles as fusions to the gene III product ofMl3 packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human PCan065. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-PCan065 antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared nucleic acid molecule encoding a variant or a non-variantversion of the anti-PCan065 antibody.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of the antibody.

Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. One mayfurther select antibodies with certain biological characteristics, asdesired.

The growth inhibitory effects of an anti-PCan065 antibody of theinvention may be assessed by methods known in the art, e.g., using cellswhich express PCan065 either endogenously or following transfection withthe PCan065 gene. For example, the tumor cell lines andPCan065-transfected cells provided in Example 1 below may be treatedwith an anti-PCan065 monoclonal antibody of the invention at variousconcentrations for a few days (e.g., 2-7) days and stained with crystalviolet or MIT or analyzed by some other colorimetric assay. Anothermethod of measuring proliferation would be by comparing ³H-thymidineuptake by the cells treated in the presence or absence an anti-PCan065antibody of the invention. After antibody treatment, the cells areharvested and the amount of radioactivity incorporated into the DNAquantitated in a scintillation counter. Appropriated positive controlsinclude treatment of a selected cell line with a growth inhibitoryantibody known to inhibit growth of that cell line. Growth inhibition oftumor cells in vivo can be determined in various ways such as isdescribed in the Experimental Examples section below. Preferably, thetumor cell is one that over-expresses PCan065. Preferably, theanti-PCan065 antibody will inhibit cell proliferation of anPCan065-expressing tumor cell in vitro or in vivo by about 25-100%compared to the untreated tumor cell, more preferably, by about 30-100%,and even more preferably by about 50-100% or 70-100%, at an antibodyconcentration of about 0.5 to 30 μg/ml. Growth inhibition can bemeasured at an antibody concentration of about 0.5 to 30 μg/ml or about0.5 nM to 200 nM in cell culture, where the growth inhibition isdetermined 1-10 days after exposure of the tumor cells to the antibody.The antibody is growth inhibitory in vivo if administration of theanti-PCan065 antibody at about 1 μg/kg to about 100 mg/kg body weightresults in reduction in tumor size or tumor cell proliferation withinabout 5 days to 3 months from the first administration of the antibody,preferably within about 5 to 30 days.

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., propidium iodide (PI), trypan blue or7AAD uptake may be assessed relative to a control. A PI uptake assay canbe performed in the absence of complement and immune effector cells.PCan065-expressing tumor cells are incubated with medium alone or mediumcontaining of the appropriate monoclonal antibody at e.g., about 10μg/ml. The cells are incubated for a 3 day time period. Following eachtreatment, cells are washed and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake may beselected as cell death-inducing antibodies.

To screen for antibodies which bind to an epitope on PCan065 bound by anantibody of interest, e.g., the PCan065 antibodies of this invention, aroutine cross-blocking assay such as that describe in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. This assay can be used to determine if atest antibody binds the same site or epitope as an anti-PCan065 antibodyof the invention. Alternatively, or additionally, epitope mapping can beperformed by methods known in the art. For example, the antibodysequence can be mutagenized such as by alanine scanning, to identifycontact residues. The mutant antibody is initially tested for bindingwith polyclonal antibody to ensure proper folding. In a differentmethod, peptides corresponding to different regions of PCan065 can beused in competition assays with the test antibodies or with a testantibody and an antibody with a characterized or known epitope.

For example, a method to screen for antibodies that bind to an epitopewhich is bound by an antibody this invention may comprise combining anPCan065-containing sample with a test antibody and an antibody of thisinvention to form a mixture, the level of PCan065 antibody bound toPCan065 in the mixture is then determined and compared to the level ofPCan065 antibody bound in the mixture to a control mixture, wherein thelevel of PCan065 antibody binding to PCan065 in the mixture as comparedto the control is indicative of the test antibody's binding to anepitope that is bound by the anti-PCan065 antibody of this invention.The level of PCan065 antibody bound to PCan065 is determined by ELISA.The control may be a positive or negative control or both. For example,the control may be a mixture of PCan065, PCan065 antibody of thisinvention and an antibody known to bind the epitope bound by the PCan065antibody of this invention. The anti-PCan065 antibody labeled with alabel such as those disclosed herein. The PCan065 may be bound to asolid support, e.g., a tissue culture plate or to beads, e.g.,SEPHAROSE® beads.

Immunoconjugates

The invention also pertains to therapy with immunoconjugates comprisingan antibody conjugated to an anti-cancer agent such as a cytotoxic agentor a growth inhibitory agent.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Conjugates of an antibodyand one or more small molecule toxins, such as a calicheamicin,maytansinoids, a trichothene, and CC1065, and the derivatives of thesetoxins that have toxin activity, are also contemplated herein.

Maytansine and Maytansinoids

Preferably, an anti-PCan065 antibody (full length or fragments) of theinvention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the cast Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DMI linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al. CancerResearch 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10 5 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansonoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Anti-PCan065 Antibody-Maytansinoid Conjugates (Immunoconjugates)

Anti-PCan065 antibody-maytansinoid conjugates are prepared by chemicallylinking an anti-PCan065 antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. An average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody, although even one molecule oftoxin/antibody would be expected to enhance cytotoxicity over the use ofnaked antibody. Maytansinoids are well known in the art and can besynthesized by known techniques or isolated from natural sources.Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.5,208,020 and in the other patents and nonpatent publications referredto hereinabove. Preferred maytansinoids are maytansinol and maytansinolanalogues modified in the aromatic ring or at other positions of themaytansinol molecule, such as various maytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B 1, andChari et al. Cancer Research 52: 127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred. Conjugates of the antibody and maytansinoid maybe made using a variety of bifunctional protein coupling agents such asN-succinimidyl(2-pyridyldithio)propionate (SPDP),succinimidyl-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such ashis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl (2-pyridyldithio)propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 [1978]) and N-succinimidyl(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. Preferably, the linkage is formedat the C-3 position of maytansinol or a maytansinol analogue.

Calicheamicin

Another immunoconjugate of interest comprises an anti-PCan065 antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I), N-acetyl- γ₁ ^(I), PSAG and θ₁ ^(I), (Hinman etal. Cancer Research 53: 3336 (1993), Lode et al. Cancer Research 5 8:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the anti-PCan065antibodies of the invention include BCNU, streptozoicin, vincristine and5-fluorouracil, the family of agents known collectively LL-E33288complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well asesperamicins (U.S. Pat. No. 5,877,296). Enzymatically active toxins andfragments thereof which can be used include diphtheria A chain, 1 5nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, for example, WO 93/21232 published Oct. 28, 1993. The presentinvention further contemplates an immunoconjugate formed between anantibody and a compound with nucleolytic activity (e.g. a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-PCan065 antibodies. Examplesinclude At²¹¹, I¹³¹, I¹²⁵, In¹¹¹, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³²,and radioactive isotopes of Lu. When the conjugate is used fordiagnosis, it may comprise a radioactive atom for scintigraphic studies,for example Tc^(99M) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc^(99M), I¹²³, In¹¹¹, Re¹⁸⁶, Re¹⁸⁸, can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such as N-succinimidyl(2-pyridyldithio)propionate (SPDP),succinimidyl(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such ashis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon labeled 1-isothiocyanatobenzyl methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al. Cancer Research 52: 127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Alternatively, a fusion protein comprising the anti-PCan065 antibody andcytotoxic agent may be made, e.g. by recombinant techniques or peptidesynthesis. The length of DNA may comprise respective regions encodingthe two portions of the conjugate either adjacent one another orseparated by a region encoding a linker peptide which does not destroythe desired properties of the conjugate.

In addition, the antibody may be conjugated to a “receptor” (suchstreptavidin) for utilization in tumor pre-targeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see W081/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form. Enzymes that are useful in themethod of this invention include, but are not limited to, alkalinephosphatase useful for converting phosphate-containing prodrugs intofree drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such asO-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; P-lactamase useful for converting drugsderivatized with P-lactams into free drugs; and penicillin amidases,such as penicillin V amidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population. The enzymes of this invention can becovalently bound to the anti-PCan065 antibodies by techniques well knownin the art such as the use of the heterobifunctional crosslinkingreagents discussed above.

Alternatively, fusion proteins comprising at least the antigen bindingregion of an antibody of the invention linked to at least a functionallyactive portion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984).

Other Antibody Modifications

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

The anti-PCan065 antibodies disclosed herein may also be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and W097/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

Vectors, Host Cells, and Recombinant Methods

The invention also provides isolated nucleic acid molecule encoding thehumanized anti-PCan065 antibody, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of theantibody. For recombinant production of the antibody, the nucleic acidmolecule encoding it is isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or inserted into a vectorin operable linkage with a promoter for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to nucleic acid molecules encoding the heavy andlight chains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

Signal Sequence Component

The anti-PCan065 antibody of this invention may be producedrecombinantly not only directly, but also as a fusion polypeptide with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the native anti-PCan065 antibody signalsequence, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders.For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, oc factor leader (includingSaccharomyces and Kluyveromyces cc-factor leaders), or acid phosphataseleader, the C. albicans glucoamylase leader, or the signal described inWO 90/13646. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available. The DNA for such precursor region is ligated inreading frame to DNA encoding the anti-PCan065 antibody.

Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theanti-PCan065 antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -11, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc. For example, cellstransformed with the DHFR selection gene are first identified byculturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti-PCan065 antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4 Jones, Genetics, 85:12 (1977). The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 pm circular plasmid pKDI canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theanti-PCan065 antibody nucleic acid. Promoters suitable for use withprokaryotic hosts include the phoA promoter, P-lactamase and lactosepromoter systems, alkaline phosphatase promoter, a tryptophan (trp)promoter system, and hybrid promoters such as the tac promoter. However,other known bacterial promoters are suitable. Promoters for use inbacterial systems also will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the anti-PCan065 antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors. Examples of suitable promoter sequences for use withyeast hosts include the promoters for 3-phosphoglycerate kinase or otherglycolytic enzymes, such as enolase, glyceraldehyde phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Anti-PCan065 antibody transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIll E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human P-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

Enhancer Element Component

Transcription of a DNA encoding the anti-PCan065 antibody of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theanti-PCan065 antibody-encoding sequence, but is preferably located at asite 5′ from the promoter.

Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-PCan065 antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO 94/11026 and the expression vectordisclosed therein.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W31 10 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation region (TIR) and signal sequences for optimizing expressionand secretion, these patents incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g., in CHOcells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-PCan065antibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-PCan065antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,Arabidopsis and tobacco can also be utilized as hosts. Cloning andexpression vectors useful in the production of proteins in plant cellculture are known to those of skill in the art. See e.g. Hiatt et al.,Nature (1989) 342: 76-78, Owen et al. (1992) Bio/Technology 10: 790-794,Artsaenko et al. (1995) The Plant J 8: 745-750, and Fecker et al. (1996)Plant Mol Biol 32: 979-986.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor (MMT060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-PCan065 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

Culturing Host Cells

The host cells used to produce the anti-PCan065 antibody of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's FIO (Sigma), Minimal Essential Medium (MEM)(Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM)(Sigma)are suitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Purification of Anti-PCan065 Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10: 163-167 (1992) describe a procedure forisolating antibodies which are secreted to the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30min. Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SIDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Pharmaceutical Formulations

Pharmaceutical formulations of the antibodies used in accordance withthe present invention are prepared for storage by mixing an antibodyhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as acetate, Tris,phosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol, and mcresol); low molecular weight(less than about 10 residues) polypeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrollidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; tonicifiers such as trehaloseand sodium chloride; sugars such as sucrose, mannitol, trehalose orsorbitol; surfactant such as polysorbate; salt-forming counter-ions suchas sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theantibody preferably comprises the antibody at a concentration of between5-200 mg/ml, preferably between 10-100 mg/ml.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, in addition to the anti-PCan065 antibody whichinternalizes, it may be desirable to include in the one formulation, anadditional antibody, e.g. a second anti-PCan065 antibody which binds adifferent epitope on PCan065, or an antibody to some other target suchas a growth factor that affects the growth of the particular cancer.Alternatively, or additionally, the composition may further comprise achemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitoryagent, anti-hormonal agent, and/or cardioprotectant. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively,in colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules) or inmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−) hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Methods and Treatment Using Anti-PCan065 Antibodies

According to the present invention, the anti-PCan065 antibody that bindsto PCan065 or internalizes upon binding to PCan065 on a cell surface isused to treat a subject in need thereof having a cancer characterized byPCan065-expressing cancer cells, in particular, ovarian, breast, colon,prostate, pancreatic or lung cancer, and associated metastases.

The cancer will generally comprise PCan065-expressing cells, such thatthe anti-PCan065 antibody is able to bind thereto. While the cancer maybe characterized by overexpression of the PCan065 molecule, the presentapplication further provides a method for treating cancer which is notconsidered to be an PCan065-overexpressing cancer.

This invention also relates to methods for detecting cells or tissueswhich overexpress PCan065 and to diagnostic kits useful in detectingcells or tissues expressing PCan065 or in detecting PCan065 in bodilyfluids from a patient. Bodily fluids include blood, serum, plasma,urine, ascites, peritoneal wash, saliva, sputum, seminal fluids, tears,mucous membrane secretions, and other bodily excretions such as stool.The methods may comprise combining a cell-containing test sample with anantibody of this invention, assaying the test sample for antibodybinding to cells in the test sample and comparing the level of antibodybinding in the test sample to the level of antibody binding in a controlsample of cells. A suitable control is, e.g., a sample of normal cellsof the same type as the test sample or a cell sample known to be free ofPCan065 overexpressing cells. A level of PCan065 binding higher thanthat of such a control sample would be indicative of the test samplecontaining cells that overexpress PCan065. Alternatively the control maybe a sample of cells known to contain cells that overexpress PCan065. Insuch a case, a level of PCan065 antibody binding in the test sample thatis similar to, or in excess of, that of the control sample would beindicative of the test sample containing cells that overexpress PCan065.

Additionally, the methods may comprise combining a test sample with anantibody of this invention, assaying the test sample for antibodybinding to PCan065 in the test sample and comparing the level ofantibody binding in the test sample to the level of antibody binding ina control sample. A suitable control is, e.g., a non-diseased sample ofthe same type as the test sample, sample known to be free of PCan065 ora sample of known quantity of PCan065. A level of PCan065 binding higherthan that of such a control sample would be indicative of the testsample containing overexpression of PCan065. Alternatively the controlmay be a sample known to overexpress PCan065. In such a case, a level ofPCan065 antibody binding in the test sample that is similar to, or inexcess of, that of the control sample would be indicative of the testsample overexpressing PCan065.

PCan065 overexpression may be detected with a various diagnostic assays.For example, over expression of PCan065 may be assayed byimmunohistochemistry (1HC). Paraffin embedded tissue sections from atumor biopsy may be subjected to the IHC assay and accorded an PCan065protein staining intensity criteria as follows.

Score 0 no staining is observed or membrane staining is observed in lessthan 10% of tumor cells.

Score 1+ a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane. Score 2+ a weak to moderate complete membrane stainingis observed in more than 10% of the tumor cells.

Score 3+ a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for PCan065 expression may becharacterized as not overexpressing PCan065, whereas those tumors with2+ or 3+ scores may be characterized as overexpressing PCan065.

Alternatively, or additionally, FISH assays such as the INFORM™ (sold byVentana, Arizona) or PATHVISION™ (VySiS, Illinois) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of PCan065 overexpression in the tumor. PCan065 overexpressionor amplification may be evaluated using an in vivo diagnostic assay,e.g. by administering a molecule (such as an antibody of this invention)which binds PCan065 and which is labeled with a detectable label (e.g. aradioactive isotope or a fluorescent label) and externally scanning thepatient for localization of the label.

A sample suspected of containing cells expressing or overexpressingPCan065 is combined with the antibodies of this invention underconditions suitable for the specific binding of the antibodies toPCan065. Binding and/or internalizing the PCan065 antibodies of thisinvention is indicative of the cells expressing PCan065. The level ofbinding may be determined and compared to a suitable control, wherein anelevated level of bound PCan065 as compared to the control is indicativeof PCan065 overexpression. The sample suspected of containing cellsoverexpressing PCan065 may be a cancer cell sample, particularly asample of ovarian, colon, prostate or lung cancer. A serum sample from asubject may also be assayed for levels of PCan065 by combining a serumsample from a subject with an PCan065 antibody of this invention,determining the level of PCan065 bound to the antibody and comparing thelevel to a control, wherein an elevated level of PCan065 in the serum ofthe patient as compared to a control is indicative of overexpression ofPCan065 by cells in the patient. The subject may have a cancer such asovarian, colon, prostate or lung cancer.

Currently, depending on the stage of the cancer, ovarian, breast, colon,prostate, pancreatic or lung cancer treatment involves one or acombination of the following therapies: surgery to remove the canceroustissue, radiation therapy, androgen deprivation (e.g., hormonaltherapy), and chemotherapy. Anti-PCan065 antibody therapy may beespecially desirable in elderly patients who do not tolerate thetoxicity and side effects of chemotherapy well, in metastatic diseasewhere radiation therapy has limited usefulness, and for the managementof prostatic carcinoma that is resistant to androgen deprivationtreatment. The tumor targeting and internalizing anti-PCan065 antibodiesof the invention are useful to alleviate PCan065-expressing cancers,e.g., ovarian, breast, colon, prostate, pancreatic or lung cancers uponinitial diagnosis of the disease or during relapse. For therapeuticapplications, the anti-PCan065 antibody can be used alone, or incombination therapy with, e.g., hormones, antiangiogens, orradiolabelled compounds, or with surgery, cryotherapy, and/orradiotherapy, notably for ovarian, breast, colon, prostate, pancreaticor lung cancers, also particularly where shed cells cannot be reached.Anti-PCan065 antibody treatment can be administered in conjunction withother forms of conventional therapy, either consecutively with, pre- orpost-conventional therapy, Chemotherapeutic drugs such as Taxotere®(docetaxel), Taxol® (paclitaxel), estramustine and mitoxantrone are usedin treating metastatic and hormone refractory ovarian, breast, colon,prostate, pancreatic or lung cancer, in particular, in good riskpatients. In the present method of the invention for treating oralleviating cancer, in particular, androgen independent and/ormetastatic ovarian, breast, colon, prostate, pancreatic or lung cancer,the cancer patient can be administered anti-PCan065 antibody inconjunction with treatment with the one or more of the precedingchemotherapeutic agents. In particular, combination therapy withpaclitaxel and modified derivatives (see, e.g., EP0600517) iscontemplated. The anti-PCan065 antibody will be administered with atherapeutically effective dose of the chemotherapeutic agent. Theanti-PCan065 antibody may also be administered in conjunction withchemotherapy to enhance the activity and efficacy of thechemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference(PDR) discloses dosages of these agents that have been used in treatmentof various cancers. The dosing regimen and dosages of theseaforementioned chemotherapeutic drugs that are therapeutically effectivewill depend on the particular cancer being treated, the extent of thedisease and other factors familiar to the physician of skill in the artand can be determined by the physician.

Particularly, an immunoconjugate comprising the anti-PCan065 antibodyconjugated with a cytotoxic agent may be administered to the patient.Preferably, the immunoconjugate bound to the PCan065 protein isinternalized by the cell, resulting in increased therapeutic efficacy ofthe immunoconjugate in killing the cancer cell to which it binds.Preferably, the cytotoxic agent targets or interferes with the nucleicacid in the cancer cell. Examples of such cytotoxic agents are describedabove and include maytansin, maytansinoids, saporin, gelonin, ricin,calicheamicin, ribonucleases and DNA endonucleases.

The anti-PCan065 antibodies or immunoconjugates are administered to ahuman patient, in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The antibodies or immunoconjugates may beinjected directly into the tumor mass. Intravenous or subcutaneousadministration of the antibody is preferred. Other therapeutic regimensmay be combined with the administration of the anti-PCan065 antibody.

The combined administration includes co-administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities. Preferably such combined therapy results in asynergistic therapeutic effect.

It may also be desirable to combine administration of the anti-PCan065antibody or antibodies, with administration of an antibody directedagainst another tumor antigen associated with the particular cancer. Assuch, this invention is also directed to an antibody “cocktail”comprising one or more antibodies of this invention and at least oneother antibody which binds another tumor antigen associated with thePCan065-expressing tumor cells. The cocktail may also compriseantibodies that are directed to other epitopes of PCan065. Preferablythe other antibodies do not interfere with the binding and orinternalization of the antibodies of this invention.

The antibody therapeutic treatment method of the present invention mayinvolve the combined administration of an anti-PCan065 antibody (orantibodies) and one or more chemotherapeutic agents or growth inhibitoryagents, including co-administration of cocktails of differentchemotherapeutic agents. Chemotherapeutic agents include, e.g.,estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil,melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such aspaclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparationand dosing schedules for such chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams & Wilkins, Baltimore, Md. (1992).

The antibody may be combined with an anti-hormonal compound; e.g., ananti-estrogen compound such as tamoxifen; an anti-progesterone such asonapristone (see, EP 616 812); or an anti-androgen such as flutamide, indosages known for such molecules. Where the cancer to be treated isandrogen independent cancer, the patient may previously have beensubjected to anti-androgen therapy and, after the cancer becomesandrogen independent, the anti-PCan065 antibody (and optionally otheragents as described herein) may be administered to the patient.

Sometimes, it may be beneficial to also co-administer a cardioprotectant(to prevent or reduce myocardial dysfunction associated with thetherapy) or one or more cytokines to the patient. In addition to theabove therapeutic regimes, the patient may be subjected to surgicalremoval of cancer cells and/or radiation therapy, before, simultaneouslywith, or post antibody therapy. Suitable dosages for any of the aboveco-administered agents are those presently used and may be lowered dueto the combined action (synergy) of the agent and anti-PCan065 antibody.

For the prevention or treatment of disease, the dosage and mode ofadministration will be chosen by the physician according to knowncriteria. The appropriate dosage of antibody will depend on the type ofdisease to be treated, as defined above, the severity and course of thedisease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Preferably, the antibody isadministered by intravenous infusion or by subcutaneous injections.Depending on the type and severity of the disease, about 1 pg/kg toabout 50 mg/kg body weight (e.g. about 0.1-15 mg/kg/dose) of antibodycan be an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. A dosing regimen can comprise administering aninitial loading dose of about 4 mg/kg, followed by a weekly maintenancedose of about 2 mg/kg of the anti-PCan065 antibody. However, otherdosage regimens may be useful. A typical daily dosage might range fromabout 1 pg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. The progress of this therapy canbe readily monitored by conventional methods and assays and based oncriteria known to the physician or other persons of skill in the art.

Aside from administration of the antibody protein to the patient, thepresent application contemplates administration of the antibody by genetherapy. Such administration of a nucleic acid molecule encoding theantibody is encompassed by the expression “administering atherapeutically effective amount of an antibody”. See, for example, WO96/07321 published Mar. 14, 1996 concerning the use of gene therapy togenerate intracellular antibodies.

There are two major approaches to introducing the nucleic acid molecule(optionally contained in a vector) into the patient's cells; in vivo andex vivo. For in vivo delivery the nucleic acid molecule is injecteddirectly into the patient, usually at the site where the antibody isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid molecule is introduced into these isolated cells and themodified cells are administered to the patient either directly or, forexample, encapsulated within porous membranes which are implanted intothe patient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). Thereare a variety of techniques available for introducing nucleic acidmolecules into viable cells. The techniques vary depending upon whetherthe nucleic acid is transferred into cultured cells in vitro, or in vivoin the cells of the intended host. Techniques suitable for the transferof nucleic acid into mammalian cells in vitro include the use ofliposomes, electroporation, microinjection, cell fusion, DEAE-dextran,the calcium phosphate precipitation method, etc. A commonly used vectorfor ex vivo delivery of the gene is a retroviral vector.

The currently preferred in vivo nucleic acid molecule transfertechniques include transfection with viral vectors (such as adenovirus,Herpes simplex I virus, or adeno-associated virus) and lipid-basedsystems (useful lipids for lipid-mediated transfer of the gene areDOTMA, DOPE and DC-Chol, for example). For review of the currently knowngene marking and gene therapy protocols see Anderson et at., Science256:808-813 (1992). See also WO 93/25673 and the references citedtherein.

Articles of Manufacture and Kits

The invention also relates to an article of manufacture containingmaterials useful for the detection of PCan065 levels in samples, PCan065overexpressing cells and/or the treatment of PCan065 expressing cancer,in particular ovarian, breast, colon, prostate, pancreatic or lungcancer. The article of manufacture comprises a container and acomposition contained therein comprising an antibody of this invention.The composition may further comprise a carrier. The article ofmanufacture may also comprise a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for detecting PCan065 expressing cells and/ortreating a cancer condition and may have a sterile access port (forexample the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an anti-PCan065 antibody of theinvention. The label or package insert indicates that the composition isused for detecting PCan065 levels, PCan065 expressing cells and/or fortreating ovarian, breast, colon, prostate, pancreatic or lung cancer, ina patient in need thereof. The label or package insert may furthercomprise instructions for administering the antibody composition to acancer patient. Additionally, the article of manufacture may furthercomprise a second container comprising a substance which detects theantibody of this invention, e.g., a second antibody which binds to theantibodies of this invention. The substance may be labeled with adetectable label such as those disclosed herein. The second containermay contain e.g., a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. The article of manufacture mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes.

Kits are also provided that are useful for various purposes, e.g., forPCan065 cell killing assays, for purification or immunoprecipitation ofPCan065 from cells or for detecting the presence of PCan065 in a bodilyfluid sample or detecting the presence of PCan065-expressing cells in acell sample. For isolation and purification of PCan065, the kit cancontain an anti-PCan065 antibody coupled to a solid support, e.g., atissue culture plate or beads (e.g., SEPHAROSE® beads). Kits can beprovided which contain the antibodies for detection and quantitation ofPCan065 in vitro, e.g. in an ELISA or a Western blot. As with thearticle of manufacture, the kit comprises a container and a compositioncontained therein comprising an antibody of this invention. The kit mayfurther comprise a label or package insert on or associated with thecontainer. The kits may comprise additional components, e.g., diluentsand buffers, substances which bind to the antibodies of this invention,e.g., a second antibody which may comprise a label such as thosedisclosed herein, e.g., a radiolabel, fluorescent label, or enzyme, orthe kit may also comprise control antibodies. The additional componentsmay be within separate containers within the kit. The label or packageinsert may provide a description of the composition as well asinstructions for the intended in vitro or diagnostic use.

EXAMPLES Example 1 Production and Isolation of Monoclonal AntibodyProducing Hybridomas

The following MAb/hybridomas of the present invention are describedbelow:

PCan065.A4, PCan065.A10, PCan065.A13, PCan065.B1, PCan065.B2,PCan065.B3, PCan065.B4, PCan065.B5, PCan065.B6, PCan065.B7, PCan065.B8,PCan065.B9, PCan065.B10, PCan065.B11, PCan065.B12, PCan065.B13,PCan065.B14, PCan065.B15, PCan065.B16, PCan065.B17, PCan065.B18,PCan065.B19, PCan065.B20, PCan065.B21, PCan065.B22, PCan065.B23,PCan065.B24, PCan065.B25, PCan065.B26, PCan065.B27, PCan065.B28,PCan065.B29, PCan065.B30, PCan065.B31, PCan065.B32, PCan065.B33,PCan065.B34, PCan065.B35, PCan065.B36, PCan065.B37, PCan065.B38,PCan065.B39, PCan065.B40, PCan065.B41, PCan065.B42, PCan065.B43,PCan065.B44, PCan065.B45, PCan065.B46, PCan065.B47, PCan065.B48,PCan065.B101, PCan065.B102, PCan065.B103, PCan065.B104, PCan065.B105,PCan065.B106, PCan065.B107, PCan065.B108, PCan065.B109, PCan065.B110,PCan065.B111, PCan065.B112, PCan065.B113, PCan065.B114, PCan065.B115,PCan065.B116, PCan065.B117 and PCan065.B118.

If the MAb producing hybridoma has been cloned, it will get thenomenclature “X#0.1,” e.g., the first clone of PCan065.A10 will bereferred to as A10.1, the second clone of A10 will be referred to asA10.2, etc. Sub-clones are designated by a subsequent “.#”, e.g. thefirst sub-clone of PCan065.A10.3 is referred to as A10.1.1, the secondsub-clone of A10.1 is A10.1.2, etc. Further generations of sub-clonesare annotated in the same format. For the purposes of this invention, areference to an anti-PCan065 antibody producing hybridoma, e.g.PCan065.A10 or A10, will include all clones and sub-clones of theantibody, e.g., A10.1, A10.2, A10.1.1, etc. Furthermore, thenomenclature PCan065.A10.3, for example, may reference the antibodyproducing hybridoma, or the antibody itself.

Immunogens and Antigens (Recombinant Proteins, His Tags)

For the PCan065 Constructs described below, nucleic acid moleculesencoding regions of PCan065 were inserted into various expressionvectors to produce recombinant proteins. These nucleic acid sequenceswere isolated by PCR using the primers which are routine to design.

For purposes of illustration, the predicted amino acid sequence encodedby each construct is also included. However, the constructs may includenaturally occurring variants (e.g. allelic variants, SNPs) within thePCan065 region as isolated by the primers. These variant sequences, andantibodies which bind to them are considered part of the invention asdescribed herein.

PCan065 Construct 1 Sequence and Protein Production

A nucleic acid molecule encoding the mature form of PCan065, Ala197 toIle308, was inserted into a modified pCMV5His2 vector at the NsiI/Nhelsites. Designing primers to isolate a nucleic acid molecule is routineto one of skill in the art.

The modified vector comprises a nucleotide sequence encoding a 17 aminoacid secretion signal sequence from human stanniocalcin 1 (STC1) plus 2transitional amino acids in frame on the 5′ side of the insertion site,and a sequence encoding 2 transitional amino acids and a 10 His tagin-frame at the 3′ side of the insertion site. The resulting vector withthe inserted PCan065 nucleic acid fragment encodes a recombinant PCan065fusion protein with the STC1 secretion signal fused to the N-terminusand the 10 His-tag fused to the C-terminus of the PCan065 proteinfragment (Ala197-Ile308). This recombinant plasmid encoding the PCan065His-tagged protein is herein referred to as “PCan065 Construct 1”. Arepresentative amino acid sequence encoded by PCn065 Construct 1 ispresented in SEQ ID NO:1. The PCan065 protein fragment (Ala197-Ile308)in PCan065 Construct 1 is located at Ala20-Ile131 of SEQ ID NO: 1.

PCan065 Construct 2 Sequence and Protein Production

A nucleic acid molecule encoding the full length of PCan065(Met1-Ile308), was inserted into a pCMV5His3 vector at the PmeI/Nhelsite. Designing primers to isolate a nucleic acid molecule is routine toone of skill in the art.

The vector comprises a sequence encoding 2 transitional amino acids anda 10 His tag in-frame at the 3′ side of the insertion site. Theresulting vector with the inserted PCan065 nucleic acid fragment encodesa recombinant PCan065 fusion protein with the His-tag fused to theC-terminus of the protein. This recombinant plasmid is herein referredto as “PCan065 Construct 2”. A representative amino acid sequenceencoded by PCan065 Construct 2 is presented in SEQ ID NO:2. The PCan065protein (Met1-Ile308) in PCan065 Construct 2 is located at Met1-Ile308of SEQ ID NO: 2.

The recombinant plasmids, PCan065 Construct 1 and PCan065 Construct 2,were used to independently transfect HEK293F cells in suspension culture(1-10 liter serum free medium) in spinner flasks. Culture medium washarvested at 48 hours post-transfection. Medium was concentrated 10-100fold, and diafiltrated with 20 mM Tris/HCl, 500 mM NaCl, 10% glycerol,pH 7.8. Concentrated medium containing protein encoded by either PCan065Construct 1 or PCan065 Construct 2 was passed through a 5-mL nickelmetal chelating column (His-Select-Ni, Sigma Inc.), which had beenpreviously equilibrated with 50 mM sodium phosphate, 1000 mM NaCl, 10%glycerol, pH 7.8. The column was then washed with 6 column volume (CV)of 50 mM sodium phosphate, 1000 mM NaCl, 20 mM imidazole, 10% glycerol,pH 7.8. Protein encoded by PCan065 Construct 1 and 2 was eluted from thecolumn using 6 CV of 50 mM sodium phosphate, 500 mM NaCl, 10% glycerol,pH 7.7 containing 500 mM imidazole. Samples from collected fractionswere subjected to SDS-PAGE and Western blot analysis for assessing thepurity of the protein. Purified fractions were pooled and dialyzedagainst PBS, pH 7.4.

The recombinant protein encoded by PCan065 Construct 1 was alternativelypurified and refolded into soluble form by the following procedure.Harvested HEK293F cells (16 grams) expressing PCan065 Construct 1 werelysed in 70 ml of 0.1 M sodium phosphate, pH 8.0, containing 0.4 M NaCl,10% glycerol and 1% Triton X-100 by sonification and centrifuged at lowspeed (100-200 RCF). The supernatant was diluted to final volume of 150ml with the same buffer and mixed with 26.5 ml of 100% ammonium sulfate(15% in final concentration). The solution was centrifuged in a BeckmanL8-70M ultracentrifuge with a Ti45 rotor at 24,000 rpm for 30 minutes at5° C. The precipitation was suspended in 25 ml of 50 mM sodium citrate,pH 5.8, containing 150 mM NaCl by sonification and centrifuged againunder the same conditions. The precipitation was dissolved in one ml of6 M guanidine/HCl and subjected to refolding.

The refolding of the purified PCan065 Construct 1 was carried out byusing the protein refolding kit from Pierce Biotechnology, Inc.(Rockford, Ill. 61105). Briefly, 50 μl of the guanidine denaturedprotein was diluted into 1 ml of each of the refolding buffers (#1-9)following the manufacture's protocol. The mixtures were stirred at 4° C.over two days. After centrifuged at 14,000 rpm for 5 minutes in aneppendorf centrifuge, the supernatants were dialyzed in 4 liters of PBS,pH 7.2, overnight at 4° C. The solutions were recovered and centrifugedagain in the same eppendorf centrifuge. The supernatants were pooled andconcentrated to 1.2 mg/ml by using a 5 kD cutoff polyethersulfonemembrane (VIVASPIN 6, VivaScience, Hannover, Germany).

Cln242 Construct 1 Sequence and Protein Production

A nucleic acid molecule encoding the full length of Cln242(Met1-Val783), was inserted into a pCMV5His2 vector at the PmeI/Nhelsite. Designing primers to isolate a nucleic acid molecule is routine toone of skill in the art.

The vector comprises a sequence encoding 6 transitional amino acids anda 10 His tag in-frame at the 3′ end of the insertion site. The resultingvector with the inserted Cln242 nucleic acid fragment encodes arecombinant Cln242 fusion protein with the 10 His-tag fused to theC-terminus of the protein. This recombinant plasmid is herein referredto as “Cln242 Construct 1”. A representative amino acid sequence encodedby Lng108 Construct 1 is presented in SEQ ID NO:3.

The recombinant plasmid Cln242 Construct 1 was used to transfect HEK293F cells in suspension culture (1-10 liter serum free medium) in abioreactor. Culture medium was harvested at 48 hours post-transfection.Medium was concentrated 10-100 fold, and diafiltrated with 20 mMTris/HCl, 500 mM NaCl, 5% glycerol, pH 7.8. Concentrated mediumcontaining protein encoded by Cln242 Construct 1 was passed through a10-mL nickel metal chelating column (His-Select-Ni, Sigma Inc.), whichhad been previously equilibrated with 50 mM sodium phosphate, 500 mMNaCl, 5% glycerol, pH 8.0. The column was then washed with 7 columnvolume (CV) of 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, 10%glycerol, pH 8.0. Protein encoded by Cln242 Construct 1 was eluted fromthe column using 9 CV of 50 mM sodium phosphate, 500 mM NaCl, 10%glycerol, pH 7.6 containing 50 mM imidazole and 10 CV of 50 mM sodiumphosphate, 500 mM NaCl, 10% glycerol, pH 7.6 containing 100 mMimidazole. Samples from collected fractions were subjected to SDS-PAGEand Western blot analysis for assessing the purity of the protein.Purified fractions were pooled and concentrated.

Cln101 Construct 1 Sequence and Protein Production

A nucleic acid molecule encoding the mature form of Cln101, Asp23 toPro158, was inserted into a modified pCMV5His2 vector at the NsiI/Nhelsites. Designing primers to isolate a nucleic acid molecule is routineto one of skill in the art.

The modified vector comprises a nucleotide sequence encoding a 23 aminoacid secretion signal sequence from human stanniocalcin 1 (STC1) plus 4transitional amino acids in frame on the 5′ side of the insertion site,and a sequence encoding 2 transitional amino acids and a 10 His tagin-frame at the 3′ side of the insertion site. The resulting vector withthe inserted Cln101 nucleic acid fragment encodes a recombinant Cln101fusion protein with the STC1 secretion signal fused to the N-terminusand the 10 His-tag fused to the C-terminus of the Cln101 proteinfragment (Asp23-Pro158). This recombinant plasmid encoding the Cln101His-tagged protein is herein referred to as “Cln101 Construct 1”. Arepresentative amino acid sequence encoded by Cln101 Construct 1 ispresented in SEQ ID NO:4.

The recombinant plasmid Cln101 Construct 1 was used to transfect HEK293F cells in suspension culture (1-10 liter serum free medium) in abioreactor. Culture medium was harvested at 48 hours post-transfection.Medium was concentrated 10-100 fold, and diafiltrated with 20 mMTris/HCl, 500 mM NaCl, 5% glycerol, pH 7.8. Concentrated mediumcontaining protein encoded by Cln101 Construct 1 was passed through a10-mL nickel metal chelating column (His-Select-Ni, Sigma Inc.), whichhad been previously equilibrated with 50 mM sodium phosphate, 500 mMNaCl, 5% glycerol, pH 8.0. The column was then washed with 7 columnvolume (CV) of 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, 10%glycerol, pH 8.0. Protein encoded by Lng108 Construct 1 was eluted fromthe column using 9 CV of 50 mM sodium phosphate, 500 mM NaCl, 10%glycerol, pH 7.6 containing 50 mM imidazole and 10 CV of 50 mM sodiumphosphate, 500 mM NaCl, 10% glycerol, pH 7.6 containing 100 mMimidazole. Samples from collected fractions were subjected to SDS-PAGEand Western blot analysis for assessing the purity of the protein.Purified fractions were pooled and concentrated.

Immunization

Eight BALB/c mice were immunized intradermally in both rear footpadswith PCan065 Construct 1 (A-series antibodies) or PCan065 Construct 2(B-series antibodies). All injections were 25 uL per foot. The firstinjection of 10 ug of antigen per mouse was in Dulbecco's phosphatebuffered saline (DPBS) mixed in equal volume to volume ratio withTitermax gold adjuvant (Sigma, Saint Louis, Miss.). Subsequently, micewere immunized twice weekly for 5 weeks. For the 2nd through 10thinjection, mice were immunized with 10 ug of antigen in 20 uL of DPBSplus 5 uL of Adju-phos adjuvant (Accurate Chemical & Scientific Corp.,Westbury, N.Y.) per mouse. The final immunization consisted of 10 ugantigen diluted in DPBS alone.

Hybridoma Fusion

Four days after the final immunization, mice were sacrificed anddraining lymph node (popliteal) tissue was collected by steriledissection. Lymph node cells were dispersed using a Tenbroeck tissuegrinder (Wheaton #347426, VWR, Brisbane, Calif.) followed by pressingthrough a sterile sieve (VWR) into DMEM and removing T-cells viaanti-CD90 (Thy1.2) coated magnetic beads (Miltenyi Biotech,Bergisch-Gladbach, Germany).

These primary B-cell enriched lymph node cells were then immortalized byelectro-cell fusion (BTX, San Diego, Calif.) with the continuous myelomacell line P3x63Ag8.653 (Kearney, J. F. et al., J. Immunology 123:1548-1550, 1979). The myeloma and B-cells were pooled at a 1:1 ratio forthe fusion. These fusion cultures were distributed at 2 million cellsper plate into wells of 96 well culture plates (Costar #3585, VWR). Theremainder of the cells was cultured in bulk in HAT selection medium for10 days and cryopreserved for future screens. Successfully fused cellswere selected by culturing in selection medium (DMEM/15% FBS) containing2.85 μM Azaserine, 50 μM Hypoxanthine (HA) (Sigma) or 50 μMHypoxanthine, 0.2 μM Aminopterin, 8 μM Thymidine (HAT) (Sigma)supplemented with recombinant human IL-6 (Sigma) at 0.5 ng/mL. Cultureswere transitioned into medium (DMEM/10% FBS) without selection and IL-6supplements for continued expansion and antibody production.

Supernatants from wells were screened by enzyme linked solid phaseimmunoassay (ELISA). Monoclonal cultures, consisting of the geneticallyuniform progeny from single cells, were established after the screeningprocedure, by sorting of single viable cells into wells of two 96 wellplates, using flow cytometry (Coulter Elite; Beckman-Coulter, Miami,Fla.). The resulting murine B-cell hybridoma cultures were expandedusing standard tissue culture techniques. Selected hybridomas werecryopreserved in fetal bovine serum (FBS) with 10% DMSO and stored inLiquid Nitrogen at −196° C. to assure maintenance of viable clonecultures.

Direct ELISA Screening & Selection of Hybridomas Producing PCan065Specific Antibodies PCan065 A-Series MAbs

Hybridoma cell lines were selected for production of PCan065 specificantibody by direct ELISA. Wells were coated with either PCan065Construct 1 or Cln242 Construct 1 as negative control. One ug/mL proteinin PBS (100 uL/well) was incubated overnight in 96 well polystyrene EIAplates (Costar #9018, VWR) at 4° C. The plate wells were washed twicewith Tris buffered saline with 0.05% Tween20, pH 7.4 (TBST). Nonspecificbinding capacity was blocked by filling the wells (300 ul/well) withTBST/0.5% bovine serum albumin (TBST/BSA) and incubating for >30 minutesat room temperature (RT). The wells were emptied and filled with 50uL/well TBST/BSA to prevent them from drying out during the samplecollection process. Hybridoma culture medium sample (50 uL) was added tothe wells and incubated for 1 hour at RT. The wells were washed 3 timeswith TBST. One hundred uL of alkaline phosphatase conjugated goatanti-mouse IgG (Fc) with minimal cross-reactivity to human Fc(P/N115-055-071, Jackson Immunoresearch), diluted 1:5000 in TBST/BSA,was added to each well and incubated for >1 hour at RT. The wells werewashed 3 times with TBST. One hundred uL of alkaline phosphatasesubstrate para-nitrophenylphosphate (pNPP) (Sigma) at 1 mg/mL in 1 MDiethanolamine buffer pH 8.9 (Pierce) was added to each well andincubated for 20 min at RT. The enzymatic reaction was quantified bymeasuring the solution's absorbance at 405 nm wavelength.

Supernatants from three hybridomas produced an absorbance value ofgreater than 0.4 in wells coated with PCan065 Construct 1 and less than0.1 in wells coated with Cln242 Construct 1, indicating specific bindingto PCan065. These hybridomas, named PCan065.A4, PCan065.A10 andPCan065.A13, were expanded and cryopreserved.

PCan065 B-Series MAbs

ELISA screens of hybridoma supernatants were performed as described forthe A-series, except that wells were coated with PCan065 Construct 2 andCln101 Construct 1. Supernatants from ninety-four hybridomas produced anabsorbance value of greater than 0.50 in wells coated with PCan065Construct 1 and less than 0.11 in wells coated with Cln101 Construct 1,indicating specific binding to PCan065. Forty eight hybridomas with thehighest signal-to-noise ratio, named PCan065.B1 to PCan065.B48, wereexpanded and cryopreserved.

Cryopreserved fusion cultures from the B-series fusion were thawed,cultured for about 2 weeks and plated at 1 cell/well into 96 well platesby single cell sorting (Beckman Coulter Elite). After about 2 weekssupernatants were screened by direct ELISA against recombinant humanGDF-15/MIC-1 (R&D Systems, Minneapolis, Minn.). Supernatants formeighteen hybridomas produced an absorbance value of greater than 1.0 inwells coated with GDF-15/MIC-1 and less than 0.1 in wells coated with anonrelevant protein, indicating specific binding to PCan065. Thesehybridomas, named PCan065.B101.1 to B118.1, were expanded andcryopreserved.

Cloning of Hybridomas Producing PCan065 Specific MAb

Based on the ELISA data above, the following hybridomas were expandedand selected for single cell cloning into 96 well culture plates by cellsorting (Coulter Elite): PCan065.A10, PCan065.B1, PCan065.B2,PCan065.B3, PCan065.B4, PCan065.B5, PCan065.B16, PCan065.B27 andPCan065.B29. After 2 weeks of culture, supernatants of each subclonewere tested by direct ELISA on wells coated with PCan065 Construct 2.Three ELISA-positive subclones per parent hybridoma were expanded andcryopreserved.

Off-Ranking Analysis of PCan065 Hybridoma Supernatants

Dissociation constants (kd) were calculated from surface plasmonresonance measurements using a BIACORE 3000 instrument (BiaCore,Piscataway, N.J.). A RAM-Fc surface was used to capture each antibodysupernatant, followed by an injection of the protein encoded by aPCan065 Construct over the captured antibody.

Flow cell 1 of a CM5 sensor chip (BiaCore) was used as a blank surfacefor reference subtractions, and was activated and then inactivated withethanolamine per standard BiaCore protocols. Flow cell 2 was used toimmobilize RAM Fc using an injection time of 12 minutes and a flow of 5ul/min. The RAM-Fc (BiaCore) was diluted to 35 ug/mL in 10 mM acetate assuggested. Standard amine coupling (BiaCore) was used to immobilize10349 RU. Hybridoma supernatants were diluted 1:2 in HBS-EP runningbuffer (BiaCore) and passed over flow cells 1 and 2. Antibodies werecaptured at 5 ul/min flow rate, 3 minute injection, and a PCan065Construct protein was injected at 5 ug/mL for 2 minutes. Thedissociation time was 3 minutes. The regeneration of the chip surface,or removal of captured hybridoma supernatants binding to the antigenbetween cycles, was performed by injecting 10 mM glycine pH 1.75 for 30seconds at 100 uL/minute.

The above procedure was performed by using the BiaCore's surfacepreparation and binding wizard included in the BiaCore control software.Results were automatically fitted using the separate ka/kd functionincluded in the BiaCore analysis software, assuming a 1:1 Langmuirbinding model. Results in Table 1a below include the antibody producinghybridoma, the response units of Mab binding (Mab RU) and antigenbinding (antigen RU), and the dissociation constant (kd).

TABLE 1a PCan065 MAb Kinetics Clone Mab RU Antigen RU kd A10.3 2413 7213.40E−04 B1.1 1832 301 2.40E−04 B2.2 1163 1528 1.12E−04 B3.1 1327 2373.66E−04 B4.1 812 357 1.73E−04 B5.3 1057 1240 2.02E−04 B16.2 727 3711.57E−04 B27.1 1308 394 2.62E−04

PCan065 MAb Isotypes

The isotypes of 2 anti-PCan065 MAbs were determined using commerciallyavailable mouse monoclonal antibody isotyping immunoassay test kits(IsoStrip, Roche Diagnostic Corp., Indianapolis, Ind.). Isotypes arelisted in Table 1b.

TABLE 1b PCan065 MAb Isotypes Clone Isotype PCan065.A10.3 IgG2b kappaPCan065.B2.2 IgG1 kappa

Direct ELISA of Purified PCan065 Specific MAb

Purified antibodies from cloned hybridomas were tested for bindingefficacy by direct ELISA against PCan065 as described above. The PCan065antibodies evaluated include PCan065.A10, PCan065.B1, PCan065.B2,PCan065.B3, PCan065.B4, PCan065.B5, PCan065.B16, PCan065.B27 andPCan065.B29. These antibodies were tested by direct ELISA on wellscoated with recombinant human GDF-15/MIC-1 from R&D Systems(Minneapolis, Minn.) at varying concentrations (listed in Table 2A and2B below). The antibodies were tested as both biotinylated andnon-biotinylated forms. Tables 2A and 2B list the absorbance for eachantibody as measured with a SpectroMax 190 instrument at 4500D.

TABLE 2A Direct ELISA with purified Anti-PCan065 MAb Concentration ofGDF-15/MIC-1 protein (ug/mL) MAb 10 5 2.5 1.25 0.625 0.313 0.156 0PCan065.A10.3 0.286 0.262 0.184 0.189 0.166 0.144 0.120 0.066PCan065.B1.1 0.057 0.054 0.067 0.054 0.056 0.054 0.051 0.054PCan065.B2.2 0.965 0.898 0.771 0.629 0.502 0.355 0.218 0.053PCan065.B3.1 0.082 0.066 0.058 0.055 0.055 0.053 0.053 0.053PCan065.B4.1 0.082 0.070 0.061 0.058 0.055 0.057 0.052 0.052PCan065.B5.3 0.055 0.052 0.051 0.053 0.054 0.052 0.055 0.052PCan065.B16.2 0.087 0.079 0.062 0.060 0.055 0.051 0.052 0.051PCan065.B27.1 0.185 0.166 0.137 0.118 0.096 0.082 0.077 0.039PCan065.B29.2 0.094 0.077 0.066 0.058 0.054 0.053 0.050 0.049

TABLE 2B Direct ELISA with purified biotinylated Anti-PCan065 MAbConcentration of GDF-15/MIC-1 protein (ug/mL) MAb 10 5 2.5 1.25 0.6250.313 0.156 0 PCan065.A10.3 2.743 2.319 1.695 1.346 1.008 0.808 0.6150.058 PCan065.B1.1 0.210 0.145 0.108 0.090 0.066 0.074 0.065 0.245PCan065.B2.2 4.000 3.789 4.000 3.138 2.149 1.679 1.100 0.069PCan065.B3.1 0.264 0.158 0.118 0.091 0.078 0.070 0.069 0.060PCan065.B4.1 0.526 0.338 0.218 0.144 0.104 0.082 0.072 0.062PCan065.B5.3 0.139 0.107 0.078 0.071 0.063 0.062 0.064 0.059PCan065.B16.2 0.511 0.361 0.231 0.172 0.120 0.088 0.073 0.058PCan065.B27.1 1.317 1.136 0.903 0.687 0.518 0.402 0.279 0.067PCan065.B29.2 0.472 0.333 0.227 0.148 0.110 0.135 0.097 0.099

PCan065 A and B-Series MAb Checkerboard ELISA

A checkerboard ELISA was ran with PCan065 antibodies above, a polyclonalGDF-15/MIC-1 antibody from R&D Systems (Minneapolis, Minn.) and amonoclonal antibody Cln248 as a negative control.

High binding polystyrene plates (Corning Life Sciences) were coatedovernight at 4° C. with 0.3 μg/well of a first anti-PCan065 MAb. Thecoating solution was aspirated off and free binding sites were blockedwith 300 μl/well Superblock-TBS (Pierce Biotechnology, Illinois) on ashaker for 1 hour at room temperature (RT). After washing 4 times withwashing buffer (TBS+0.05% Tween20), 80 μl of Assay Buffer (TBS, 1% BSA,1% Mouse Serum, 1% Calf Serum, and 0.1% ProClin™) was added to eachwell, followed by 20 μl of antigen per well. The plate was incubated for60 minutes on a shaker. For each sandwich ELISA, standards of specifiedconcentrations of recombinant human mature form GDF-15/MIC-1 (R&DSystems, Minneapolis, Minn.) were run in parallel with test samples.Standards and test samples were diluted in Assay Buffer. For detection,100 μl of a second biotinylated MAb (0.15 μg/ml) was added to each welland incubated for 1 hour at room temperature (RT), while shaking. Afterwashing, 100 μL of Streptavidin-HRP conjugate (Jackson Lab) at 1:80,000dilution in TBS, was added to each well. Plates were then incubated withshaking at RT for 30 min. After washing the plate, 100 uL/well ofTMB-Stable Stop substrate (Moss, Inc.) was added to each well and theplate was incubated at RT, covered and on the shaker for 15 minutes. Thereaction was stopped using 100 μl/well 1N HCl, and the plates were readat 450 nm using a Spectramax 190 plate reader (Molecular Devices).

PCan065 A and B-Series MAb ELISA Pairing Results

The results of the checkerboard ELISAs on anti-PCan065 MAbs are shown inTables 3a and 3b below. Each antibody was tested as both a coating anddetecting antibody, in all possible combinations. All pairs were testedwith 10 ng/ml of recombinant human GDF-15/MIC-1 (mature form) in bufferor with buffer alone as a blank (negative control). A non binding Cln248MAb was also used as a negative control. The results in table 3a beloware shown as specific signal/noise ratio. Capturing MAbs are listed onthe Y-axis with detecting MAbs on the X-axis.

TABLE 3a Pairing of PCan065 A and B-series MAb by Sandwich ELISAdetecting 2* MAb GDF15 Cln248 A10.3 B1.1 B2.2 B3.1 B4.1 B5.3 B16.2 B27.1B29.2 Poly MAb coating 1* MAb A10.3 1 1 18 2 2 1 2 1 1 18 1 B1.1 1 1 1 11 1 1 1 1 1 1 B2.2 25 1 3 1 2 1 2 20 2 11 1 B3.1 1 1 3 1 1 1 1 1 1 8 1B4.1 4 1 2 1 1 4 2 1 3 3 1 B5.3 1 1 1 1 1 1 1 1 1 2 2 B16.2 3 1 2 1 1 11 2 1 2 1 B27.1 5 1 61 10 4 2 5 1 5 9 2 B29.2 2 1 3 1 1 1 1 2 1 2 1Cln248 1 1 1 1 1 1 1 1 1 1 1

In a second checkerboard ELISA additional anti-PCan065 MAbs wereevaluated for pairing as above. Results in Table 3b below are shown as aspecific signal/noise ratio. Capturing MAbs are listed on the Y-axiswith detecting MAbs on the X-axis.

TABLE 3a Pairing of PCan065 A and B-series MAb by Sandwich ELISAdetecting 2* Mab B2.2 B102 B111 A10.1 B101 B104 B106 B108 B114 B27.1B110 coating 1* Mab B2.2 3 6 4 15 5 8 9 29 8 22 4 B102 2 2 1 19 5 33 3622 34 2 5 B111 3 1 2 29 11 37 54 34 52 18 11 A10.1 50 50 49 2 2 2 2 2 22 2 B101 71 81 76 1 1 1 1 1 1 1 1 B104 42 50 52 1 1 1 1 1 1 1 1 B106 7664 59 1 1 2 1 2 1 1 1 B108 45 26 31 1 1 1 1 1 1 1 1 B114 75 75 71 1 1 11 1 1 1 1 B27.1 76 74 58 1 1 2 1 1 2 1 1 B110 29 27 18 1 1 1 1 1 1 1 1B112 81 54 52 1 1 2 1 2 1 1 1 B113 11 13 10 1 1 1 1 1 1 1 1 B115 80 6767 1 1 2 1 1 1 1 1 B116 43 48 39 1 1 1 1 1 1 1 1 B3.1 5 5 5 1 1 1 1 1 11 1 B1.1 1 1 1 1 1 1 1 1 1 1 1 B4.1 2 2 1 2 1 1 1 2 1 1 1 B5.3 1 1 2 1 11 1 1 1 1 1 B16.2 2 2 1 1 1 1 1 1 1 1 1 B29.2 3 4 3 1 1 1 1 1 1 1 1Cln248 1 1 1 1 1 1 1 1 1 1 1 detecting 2* Mab B113 B115 B116 B3.1 B1.1B4.1 B5.3 B16.2 B29.2 Cln248 coating 1* Mab B2.2 4 4 3 1 1 2 1 3 5 1B102 5 4 4 1 1 1 1 1 1 1 B111 10 10 7 1 1 1 1 2 2 1 A10.1 2 2 2 3 2 2 22 3 2 B101 2 1 1 3 1 1 1 1 1 1 B104 1 1 1 2 1 1 1 1 2 1 B106 2 1 1 2 1 21 2 2 1 B108 1 1 1 1 1 4 1 3 3 1 B114 1 1 1 2 1 2 1 2 1 1 B27.1 2 1 1 71 3 2 3 2 1 B110 1 1 1 2 1 1 1 1 1 1 B112 2 1 1 3 1 2 1 2 2 1 B113 1 1 11 1 1 1 1 1 1 B115 2 1 1 3 1 1 1 1 1 1 B116 1 1 1 2 1 1 1 1 1 1 B3.1 1 11 1 1 1 1 1 1 1 B1.1 1 1 1 1 1 1 1 1 1 1 B4.1 1 1 1 1 1 1 1 1 1 1 B5.3 11 1 1 1 1 1 1 1 1 B16.2 1 1 1 1 1 1 1 1 1 1 B29.2 1 1 1 1 1 1 1 1 1 1Cln248 1 1 1 1 1 1 1 1 1 1

The signal/noise ratios recorded are relative to each ELISA experiment.Therefore, data in Tables 3a and 3b demonstrate which MAbs pair well todetect PCan065 compared to others MAb pairs evaluated. Absolute valuesshould not be compared between Tables 3a and 3b. Results from the ELISApairing demonstrate that the anti-PCan065 MAbs detect several distinctepitopes. The GDF-15 polyclonal antibody from R&D Systems bound GDF-15indiscriminately in the assays represented in the checkerboard. Incontrast, monoclonal antibodies PCan065.A10, PCan065.B1, PCan065.B2,PCan065.B3, PCan065.B4, PCan065.B5, PCan065.B16, PCan065.B27 andPCan065.B29 had specific binding patterns in the assays represented inthe checkerboard, demonstrating unique binding epitopes. An epitope mapof the PCan065 MAbs derived from the pairing results is shown in FIG. 1.Antibodies PCan065.B1 and PCan065.B5 detected the full length GDF-15protein. Antibody pairs with the highest signal/noise ratio wereselected to test sensitivity for recombinant protein, reactivity towardsnative protein in cell lines and serum samples.

PCan065 Sandwich ELISA Format and Standard Curve

In the screening and pairing assays above, anti-PCan065 antibody pair(capture/detect) A10.3/B2.2 demonstrated high specificity and excellentsensitivity for the detection of PCan065. These antibody pairs wereselected for use in a sandwich ELISA for detection of PCan065.

For the A10.3/B2.2 ELISA pair GDF15/MIC-1 (R&D Systems) standards wererun at concentrations of 6, 2, 0.75, 0.3, 0.05, and 0 ng/ml in parallelwith the samples. A sensitive detection system based on the use ofhorseradish peroxidase (HRP) (1:80,000 dilution) and high sensitivityTMB-Stable Stop substrate (MOSS) was used evaluations below. Analternative assay format with a sensitive alkaline phosphatase (AP)detection system may be used. The AP system is based on the use ofalkaline phosphatase and p-Nitrophenyl phosphate (pNNP) substrate. Ineither assay format the development enzyme (HRP or AP) may be directlyconjugated to the B2.2 detection antibody for simplicity of the ELISAprocedure.

The minimal detectable dose (MDD) for PCan065 in the ELISA format,A10.3/B2.2, was determined to be 0.015 ng/ml. For calculation of medianvalues, samples with values below the MDD were defined as MDD. The MDDis defined as two standard deviations above the background signal. Astandard curve for the A10.3/B2.2 assay format is depicted in FIG. 2.This assay can accurately quantify PCan065 (MIC-1) in the range of about0.015-6 ng/mL. This assay has a greater detection range and lower endsensitivity than other MIC-1 ELISAs, which have a quantification rage ofabout 20-900 pg/mL or 0.020-0.9 ng/mL (see Moore et al. J ClinEndocrinol Metab. 2000 December; 85(12):4781-8).

Example 2 Monoclonal Sandwich ELISA Detection of PCan065 in Human SerumSamples Human Serum Samples

Human cancer and benign serum samples were obtained from IMPATH-BCP,Inc. (Franklin, Mass.), Diagnostic Support Services, Inc. (WestYarmouth, Mass.) and ProteoGenex (Culver City, Calif.). The serumsamples from healthy men and women were obtained from ProMedDx LLC(Norton, Mass.). All samples were aliquoted upon arrival and stored atminus 80° C. until use.

The concentration of PCan065 was measured in more than 2228 serumsamples from normal/healthy individuals, individuals with lung, breast,colon, prostate or ovarian cancer and individuals with non-cancerous,benign diseases. Benign diseases are grouped by tissue type and include:A. Hyperplasia, Fibroadenoma, and Fibrocystic Breasts for Breast;Crohn's, Diverticulitis, Ulcerative Colitis, and Polyps for Colon;Asthma, Chronic Bronchitis, Emphysema, Interstitial Lung Disease, andPulmonary Hypertension for Lung; Endometriosis, Enlarged Ovaries, andPolycystic Ovaries for Ovarian; Benign Prostatic Hyperplasia, ProstaticIntraepithelial Neoplasia, and Prostatitis for Prostate. An overview ofall samples tested is listed in the table 4 below.

TABLE 4 Summary of serum samples: Sample Type No. of Samples Tested No.of Samples in Analysis Normal 594 (285-F, 309-M) 565 (274-F, 291-M)Breast Cancer 150 145 Breast Benign 180 168 Colon Cancer 125 122 ColonBenign 60 56 Lung Cancer 198 191 Lung Benign 248 239 Ovarian Cancer 9797 Ovarian Benign 276 261 Prostate Cancer 156 152 Prostate Benign 144140

It has been previously shown that a polymorphism in the MIC-1 genealters the histidine to an aspartic acid (MIC-1 H and MIC-1 D) atposition 6 of the mature protein. See Brown et al., Biotechniques. 2002July; 33(1):118-20, 122, 124 passim A small portion (4%) of the samplepopulation in Table 4 was undetectable by the PCan065 A10.3/B2.2 ELISAwhich is in agreement with the differing phenotypes resulting for thispolymorphism. Only samples with PCan065 values above the ELISA MDD(0.015 ng/mL) are reported and evaluated below.

Detection of PCan065 in Serum with Sandwich ELISAs

In the following tables demonstrating detection of PCan065 in serum,samples are grouped by type and identified by tissue and disease stateof the tissue. Tissue annotation includes: BR=Breast, CN=Colon, LN=Lung,OV=Ovarian, and PR=Prostate. Disease states may be specificallyindicated or abbreviated into groups as: CAN=Cancer and BEN=Benign.Samples from non-diseased men and women are annotated as NRM Male (NRMM) and NRM Female (NRM F), respectively. For example, BR CAN indicatesbreast cancer samples and CN BEN indicates benign colon disease samples.

Benign Diseases are abbreviated as: A. Hyperplasia (AHYP), Fibroadenoma(FBAD), Fibrocystic Breasts (FBCY), Crohn's (CHRN), Diverticulitis(DVCT), Ulcerative Colitis (UCOL), Polyps (PLYP), Asthma (ASMA), ChronicBronchitis (CBRN), Emphysema (EMPH), Interstitial Lung Disease (MD),Pulmonary Hypertension (PLHP), Benign Tumor/Cyst (BTC), Cystadenofibroma(CADF), Cystadenoma (CAD), Endometriosis (ENDO), Enlarged Ovaries(ENOV), Polycystic Ovaries (PCYS), Benign Prostatic Hyperplasia (BPH),Prostatic Intraepithelial Neoplasia (PIN), and Prostatitis (PRST).

PCan065 A10.3/B2.2 MAb ELISA Results

The concentration of PCan065 in serum from 594 healthy individuals, 726individuals with cancer and 908 individuals with benign disease wasdetermined with the PCan065 A10.3/B2.2 MAb ELISA. Of the 2228 samplestested, 92 samples (4%) were undetectable with the A10.3/B2.2 assay.These 92 samples are excluded in the analyses shown below, actual numberof samples included is shown in Table 5.

Table 5 below shows the number of samples tested in each group ofindividuals, the minimum and maximum detected PCan065 concentration, themedian PCan065 concentration, and the 25^(th) and 75^(th) percentileconcentration of PCan065 in each group. Elevated levels of PCan065 wereobserved in individuals with breast, colon, lung, ovarian and prostatecancer.

TABLE 5 PCan065 Levels (ng/mL) in Normal and Cancer Samples (A10.3/B2.2ELISA) NML F NML M BR CAN CN CAN LN CAN OV CAN PR CAN Number of values274 291 145 122 191 97 152 Minimum (ng/mL) 0.115 0.018 0.016 0.061 0.0160.021 0.017 25th Percentile (ng/mL) 0.312 0.272 0.613 0.448 0.983 0.6850.535 Median (ng/mL) 0.543 0.514 1.265 0.818 1.710 1.304 0.954 75thPercentile (ng/mL) 0.895 0.748 1.784 1.409 3.007 2.059 1.686 Maximum(ng/mL) 3.733 1.885 17.170 10.060 10.690 13.820 6.853

The concentration of PCan065 was also measured in serum samples fromindividuals with various benign diseases with the PCan065 A10.3/B2.2 MAbELISA. Tables 6A, 6B, 6C, 6D, 6E and 6F below show the number of samplestested in each group (listed above), the minimum and maximum detectedPCan065 concentration, the median PCan065 concentration, and the 25^(th)and 75^(th) percentile concentration of PCan065 in each group.

TABLE 6A PCan065 Levels (ng/mL) in Breast Cancer and Benign Samples(A10.3/B2.2 ELISA) BR CAN BR BEN no All All BR stage stage stage BR NRMF CAN 1/2 3/4 info BEN AHYP FBAD FBCY Samples 274 145 100 41 4 168 52 5660 Minimum 0.115 0.016 0.016 0.228 0.242 0.021 0.178 0.110 0.021 25^(th)Percentile 0.312 0.613 0.579 0.785 0.362 0.341 0.401 0.322 0.426 Median0.543 1.265 1.224 1.433 0.542 0.795 0.809 0.703 0.902 75^(th) Percentile0.895 1.784 1.747 1.878 1.397 1.157 0.995 1.151 1.248 Maximum 3.73317.170 17.170 7.572 2.193 6.583 1.995 3.719 6.583

TABLE 6B PCan065 Levels (ng/mL) in Colon Cancer and Benign Samples(A10.3/B2.2 ELISA) CN BEN CN CAN All All CN stage stage CN NRM F NRM MCAN 1/2 3/4 BEN CHRN PLYP UCOL Samples 274 291 122 109 13 56 3 51 2Minimum 0.115 0.018 0.061 0.061 0.492 0.113 0.248 0.113 0.876 25^(h)Percentile 0.312 0.272 0.448 0.420 0.512 0.288 n/a 0.284 n/a Median0.543 0.514 0.818 0.802 1.008 0.514 0.463 0.510 0.993 75^(th) Percentile0.895 0.748 1.409 1.389 1.929 0.855 n/a 0.831 n/a Maximum 3.733 1.88510.060 10.060 6.453 4.762 1.166 4.762 1.109

TABLE 6C PCan065 Levels (ng/mL) in Lung Cancer and Benign Samples(A10.3/B2.2 ELISA) LN CAN LN BEN All LN stage stage All LN NRM F NRM MCAN 1/2 3/4 BEN ASMA CBRN EMPH ILD PLHP Samples 274 291 191 74 117 23946 47 49 48 49 Minimum 0.115 0.018 0.016 0.016 0.017 0.149 0.175 0.1490.226 0.156 0.231 25^(th) Percentile 0.312 0.272 0.983 0.882 1.101 0.7750.659 0.653 0.736 0.761 1.291 Median 0.543 0.514 1.710 1.716 1.686 1.4990.991 1.169 1.609 1.717 2.364 75^(th) Percentile 0.895 0.748 3.007 2.9873.064 2.456 1.790 2.038 2.151 3.008 4.256 Maximum 3.733 1.885 10.69010.690 10.690 14.590 14.590 5.288 10.620 5.841 11.210

TABLE 7D PCan065 Levels(ng/mL) in Ovarian Cancer and Benign Samples(A10.3/B2.2 ELISA) OV CAN OV BEN All OV stage stage All OV NRM F CAN 1/23/4 BEN BTC CADF CAD ENDO ENOV PCYS Samples 274 97 51 46 261 22 5 36 9450 54 Minimum 0.115 0.021 0.222 0.021 0.027 0.178 0.323 0.156 0.0270.093 0.035 25^(th) 0.312 0.685 0.486 0.854 0.231 0.474 0.360 0.2760.192 0.183 0.185 Percentile Median 0.543 1.304 1.138 1.468 0.409 1.1680.578 0.505 0.384 0.280 0.475 75^(th) 0.895 2.059 1.732 2.371 0.6623.227 1.445 0.671 0.645 0.467 0.617 Percentile Maximum 3.733 13.82013.820 5.943 12.630 11.030 2.124 1.132 12.630 6.658 5.619

TABLE 7E PCan065 Levels(ng/mL) in Ovarian Cancer Subtypes (A10.3/B2.2ELISA) Mucinous OV CAN Serous OV CAN All stage stage All stage stage NRMF Mucinous 1/2 3/4 Serous 1/2 3/4 Samples 274 31 19 12 66 32 34 Minimum0.115 0.021 0.222 0.021 0.038 0.341 0.038 25th Percentile 0.312 0.4490.449 0.961 0.701 0.631 0.854 Median 0.543 1.299 1.119 2.014 1.317 1.2131.418 75th Percentile 0.895 2.098 1.438 2.873 2.136 1.767 2.303 Maximum3.733 5.339 3.471 5.339 13.820 13.820 5.943

TABLE 7F PCan065 Levels(ng/mL) in Prostate Cancer and Benign Samples(A10.3/B2.2 ELISA) PR CAN All PR BEN PR stage stage no stage All BR NRMM CAN 1/2 3/4 info BEN BPH PIN PRST Samples 291 152 112 37 3 140 62 1068 Minimum 0.018 0.017 0.067 0.017 1.207 0.017 0.018 0.079 0.017 25^(th)Percentile 0.272 0.535 0.568 0.393 n/a 0.414 0.406 0.394 0.407 Median0.514 0.954 0.952 0.937 1.779 0.901 0.943 1.356 0.784 75^(th) Percentile0.748 1.686 1.697 1.603 n/a 1.421 1.842 1.748 1.207 Maximum 1.885 6.8536.853 4.697 2.229 8.886 8.886 2.671 4.003

Elevated levels of PCan065 were observed in individuals with breast,colon, lung, ovarian, and prostate cancer. PCan065 was elevated in earlystage (1/2) mucinous and serous ovarian cancer. PCan065 levels were notelevated significantly in individuals with breast, colon and ovarianbenign conditions. These results demonstrate that elevated levels ofPCan065 is indicative of an individual having breast, colon, lung,ovarian, or prostate cancer. Further, elevated levels of PCan065 areindicative early stage (stage 1/2) mucinous or serous ovarian cancer.Additionally, a PCan065 ELISA is able to determine PCan065 levels anddiscriminate individuals with breast, colon, lung, ovarian, and prostatecancers from individuals without disease and/or individuals with benigndiseases.

Example 3 ROC Analysis of PCan065 Levels in Serum

The ability of a test or assay to discriminate diseased cases fromnormal cases is evaluated using Receiver Operating Characteristic (ROC)curve analysis (Metz, 1978; Zweig & Campbell, 1993). ROC curves can alsobe used to compare the diagnostic performance of two or more laboratoryor diagnostic tests (Griner et al., 1981).

An ROC curve is generated by plotting the sensitivity against thespecificity for each value. From the plot, the area under the curve(AUC) can be determined. The value for the area under the ROC curve(AUC) can be interpreted as follows: an area of 0.84, for example, meansthat a randomly selected positive result has a test value larger thanthat for a randomly chosen negative result 84% of the time (Zweig &Campbell, 1993). When the variable under study can not distinguishbetween two result groups, i.e. where there is no difference between thetwo distributions, the area will be equal to 0.5 (the ROC curve willcoincide with the diagonal). When there is a perfect separation of thevalues of the two groups, i.e. there no overlapping of thedistributions, the area under the ROC curve equals 1 (the ROC curve willreach the upper left corner of the plot).

The 95% confidence interval for the area can be used to test thehypothesis that the theoretical area is 0.5. If the confidence intervaldoes not include the 0.5 value, then there is evidence that thelaboratory test has the ability to distinguish between the two groups(Hanley & McNeil, 1982; Zweig & Campbell, 1993).

ROC Analysis of PCan065 A10.3/B2.2 MAb ELISA

PCan065 A10.3/B2.2 MAb ELISAs sensitivity and specificity for detectingcancer was calculated through receiver operating characteristic (ROC)analysis. Table 8 below shows the results of the Area Under the Curve(AUC) from the ROC analysis for PCan065 levels in case (cancer samples)versus controls (normal healthy samples and benign disease samples fromthe corresponding organ). AUC values were calculated using the PCan065concentration levels described above with the A10.3/132.2 MAb ELISA.

TABLE 8 PCan065 (A10.3/B2.2 MAb ELISA) AUC Values for Various CancersCases (N) Controls (N) AUC 95^(th) Confidence Interval Breast 145 4420.713 0.674 to 0.749 Colon 122 621 0.666 0.631 to 0.700 Lung 191 8040.784 0.757 to 0.809 Ovarian 97 535 0.791 0.758 to 0.822 Prostate 152431 0.679 0.639 to 0.716

ROC Analysis of PCan065 CA125

The sensitivity and specificity for PCan065 and CA125 alone or incombination to detect ovarian cancer was calculated through receiveroperating characteristic (ROC) analysis. Table 9 below shows the resultsof the Area Under the Curve (AUC) from the ROC analysis for PCan065 andCA125 levels in cases (cancer samples) versus controls (normal healthysamples and benign disease samples from the corresponding organ) in allovarian cancer samples, early stage (stage 1/2) samples and late stage(stage 3/4) samples. AUC values were calculated using PCan065 levelsdescribed above and CA125 levels determined by Lumipulse from FujirebioInc. (Tokyo, Japan) in a set of the samples described above.

TABLE 9 PCan065 and CA125 AUC Values for Ovarian Cancer AUC CasesControls PCan065 + (N) (N) PCan065 CA125 CA125 All Ovarian 87 308 0.8040.774 0.835 Cancer Early Stage OvCa 46 308 0.787 0.724 0.802 Late StageOvCa 41 308 0.822 0.829 0.872

Results of PCan065 ROC Analyses

The results from the ROC analyses of the PCan065 ELISAs demonstrate thatPCan065 alone is useful for detecting cancer. PCan065 performs betterthan the established marker CA125 for detection of ovarian cancer,specifically in detecting early stage (stage 1/2) cancers. Furthermore,PCan065 and CA125 in combination have a higher AUC for detecting ovariancancers, including early stage, than either marker alone. These resultsdemonstrate PCan065, alone or in combination with other markers, isuseful for detecting cancer, in particular ovarian cancer.

Example 4 Deposits Deposit of Cell Lines and DNA

The following hybridoma cell lines were deposited with the American TypeCulture Collection (ATCC) located at 10801 University Boulevard,Manassas, Va. 20110-2209, U.S.A., and accorded accession numbers.

TABLE 10 ATCC deposits Hybridoma ATCC Accession No. Deposit DatePCan065.A10.3.2 PCan065.B2.2.1

The names of the deposited hybridoma cell lines above may be shortenedfor convenience of reference. E.g. A10.3 corresponds to PCan065.A10.3.These hybridomas correspond to the clones (with their full names) listedin Table 10. Subclones of hybridomas are listed which have the samecharacteristics and properties of parental clones. Reference to a parentclone or hybridoma producing an anti-PCan065 antibody, such asPCan065.A10 or PCan065.B2, includes all subclones such as those listedin Table 10 above.

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations there under (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of deposit. The organisms will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweendiaDexus, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the cultures to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC§122 and the Commissioner's rules pursuant thereto (including 37 CFR§1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrains are not to be construed as a license to practice the inventionin contravention of the rights granted under the authority of anygovernment in accordance with its patent laws. The making of thesedeposits is by no means an admission that deposits are required toenable the invention

1-78. (canceled)
 79. A kit for detection and quantitation of PCan065comprising an anti-PCan065 antibody, wherein PCan065 can be quantifiedin the range of about 0.015-6 ng/mL with the kit.
 80. The kit of claim79 wherein the antibody is a polyclonal, monoclonal, humanized, or humanantibody.
 81. The kit of claim 79 wherein the antibody is detectablylabeled.
 82. The kit of claim 81 wherein the label is selected from thegroup comprising fluorescent, radio and enzymatic labels.
 83. The kit ofclaim 79 further comprising diluents or buffers.
 84. The kit of claim 79further comprising a package insert.
 85. The kit of claim 84 wherein thepackage insert indicates the kit is used for detecting PCan065 levels.86. The kit of claim 84 wherein the package insert provides instructionsfor the intended in-vitro or diagnostic use of the kit.